Information

Protocol to dilute DNA step ladder?

Protocol to dilute DNA step ladder?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I need to run gels that are of not the "most" importance. So I do not want to waste alot of money on step ladder. How do I dilute DNA step ladder? Is there a general procedure, for I have tried googling for one, but I get varying answers, such as a 1:4 dilution, and so on.

This is the information on the Step ladder, by Promega, posted as an image, since copy and pasting would mess up readability.

EDIT:

Even though I awarded the bounty because it gave me the same concept; what I had actually did was to assume I would use $60ul$ of ladder combined with $1$x dye.

Therefore since I started with $1ml$ of $6$x loading dye, $100ul$ of DNA step ladder, and available nuclease free $ce{H2O}$, and I desired a 5:1 ratio of solution(dye and $ce{H2O}$) to ladder.

I would then use $12ul$ of stock to the $60ul$ total.

To make the dye $1$x, I would add $10ul$ of the $6$x dye to make the final concenetration of dye $1$x.

Then I would add up to $38ul$ with nuclease free $ce{H2O}$.

So, sorry if this was worded redundantly, but It helps me to read, therefore in summary:

$$ +10ul ext{ of 6x dye}$$ $$ +12ul ext{ of stock 100bp DNA step Ladder}$$ $$+ 38ul ext{ Nuclease Free } ce{H2O} $$ $$ ext{__________________________________}$$

$$sum{60ul} ext{ of ladder & dye} (1 ext{x}) $$

of which total I can aliquot out to my hearts desire.

EDIT2: The ladder turned out successful.


You'll probably have to titrate it down yourself. You might be able to estimate: The detection limit for ethidium bromide staining is about $1 ng$ per band. The insert says it's at "1 $mu g/ mu l$", but that's for all 40 bands and they're clearly not all at the same concentration. Depending on which bands you care about (1600 and below are dim), you might be able to get away with $(frac{1}{25})^{th}$ their recommended concentration (1 $mu g / 40$ bands = 25 $ng$) -- but I would titrate a small amount and see what works.


Protocol for DNA Cleanup and Concentration Using the Monarch® PCR & DNA Cleanup Kit (5 &mug) (NEB #T1030)

Important Update: Beginning in May 2021, we will be gradually transitioning the Monarch DNA Cleanup Binding Buffer to a concentrated format which requires the addition of isopropanol by the user. The protocol below has been updated to reflect this change, but please refer to the instructions provided with your products, as your lot may not be affected.

There are two protocols available for this product:

    DNA Cleanup and Concentration (below): for the purification of up to 5 &mug of DNA (ssDNA > 200 nt and dsDNA > 50 bp) from PCR and other enzymatic reactions.


Ordering information

We supply a wide range of Invitrogen™ DNA ladders and markers for accurate size and mass estimation (quantitation) of DNA fragments. DNA ladders and markers are available for sizing double-stranded, single-stranded, or supercoiled DNA.

A variety of these DNA ladders and markers are also available in the ready-to-load TrackIt™ format. There is no need to heat, mix, or dilute these markers prior to loading them on your gel. TrackIt™ markers contain two tracking dyes that indicate when maximum resolution of the DNA fragments has been achieved.

Resources

Nucleic acid education

Learn about gel electrophoresis basics, workflows, considerations, applications, and troubleshooting in separation and analysis of nucleic acids.


Protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥100 ng

Starting Material: 100&ndash500 ng purified, genomic DNA. We recommend that the DNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5&ndash8, low EDTA TE or H2O are also acceptable. If the input DNA is less than 26 µl, add TE (provided) to a final volume of 26 µl.

2.1. Fragmentation/End Prep
Fragmentation occurs during the 37°C incubation step. Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. See Figure 2.1 for a typical fragmentation pattern.

FRAGMENTATION SIZE
INCUBATION @ 37°C
OPTIMIZATION
100 bp-250 bp
30 min
30-40 min
150 bp-350 bp
20 min
20-30 min
200 bp-450 bp
15 min
15-20 min
300 bp-700 bp
10 min
5-15 min
500 bp-1 kb 5 min 5-10 min

Figure 2.1: Example of size distribution on a Bioanalyzer®. Human DNA (NA19240) was fragmented for 5-40 min.

2.1.1. Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.

2.1.2. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.

Note: It is important to vortex the enzyme mix prior to use for optimal performance.

2.1.3. Add the following components to a 0.2 ml thin wall PCR tube on ice:

COMPONENT
VOLUME PER ONE LIBRARY
DNA 26 µl
(yellow) NEBNext Ultra II FS Reaction Buffer
7 µl
(yellow) NEBNext Ultra II FS Enzyme Mix
2 µl
Total Volume
35 µl

2.1.4. Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.

2.1.5. In a Thermocycler, with the heated lid set to 75°C, run the following program:

5&ndash30 min @ 37°C
30 min @ 65°C
Hold @ 4°C

If necessary, samples can be stored at &ndash20°C however, a slight loss in yield (

20%) may be observed. We recommend continuing with adaptor ligation before stopping.

2.2.1. Add the following components directly to the FS Reaction Mixture:

COMPONENT VOLUME
FS Reaction Mixture (Step 2.1.5.) 35 µl
(red) NEBNext Ultra II Ligation Master Mix*
30 µl
(red) NEBNext Ligation Enhancer
1 µl
(red) NEBNext Adaptor for Illumina**
2.5 µl
Total Volume 68.5 µl

* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.

** The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535 and #E6609) Oligos for Illumina.

Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend adding adaptor to a premix in the Adaptor Ligation Step.

2.2.2. Set a 100 µl or 200 µl pipette to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. (Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).

2.2.3. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid off.

2.2.4. Add 3 µl of (red) USER ® Enzyme to the ligation mixture from Step 2.2.3.

Note: Steps 2.2.4. and 2.2.5. are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535 and #E6609) Oligos for Illumina.

2.2.5. Mix well and incubate at 37°C for 15 minutes with the heated lid set to &ge 47°C.

Samples can be stored overnight at -20°C.

2.3. Size Selection of Adaptor-ligated DNA for DNA Input > 100 ng.

If the starting material is > 100 ng, follow the protocol for size selection below. For Inputs < 100 ng, size selection is not recommended. Follow the protocol for cleanup without size selection in Chapter 1 Section 1.3. If you want fragment sizes > 550 bp and your input is > 100 follow the entire protocol in Chapter 3


Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step (71.5 µl Step 2.2.5.). These volumes may not work properly for a size selection at a different step in the workflow, or if this is a second size selection. For size selection of samples contained in different buffer conditions bead volumes may need to be experimentally determined.


The following size selection protocol is for libraries with 150-200 bp inserts only. For libraries with different size fragment inserts, refer to Table 2.3.1. below for the appropriate volumes of beads to be added. The size selection protocol is based on a starting volume of 100 µl. Size selection conditions were optimized with SPRIselect or NEBNext Sample Purification Beads however, AMPure XP beads can be used following the same conditions. If using AMPure XP beads, please allow the beads to warm to room temperature for at least 30 minutes before use.

To select a different insert size than 200 bp, please use the volumes in this table:

Table 2.3.1: Recommended conditions for bead based size selection.

APPROXIMATE
INSERT SIZE DISTRIBUTION
150-250 bp 200-350 bp 275-475 bp 350-600 bp
LIBRARY
PARAMETERS
Approx. Final Library Size Distribution (Insert+Adaptor+primers)
270-370 bp 320-470 bp
400-600 bp
470-800 bp
VOLUME TO
BE ADDED (µl)
1st Bead Selection
40 30 25 20
2nd Bead Selection
20 15 10 10

2.3.1. Bring the volume of the reaction up to 100 µl by adding 28.5 µl 0.1X TE (dilute 1X TE Buffer 1:10 with water).

2.3.2. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

2.3.3. Add 40 &mul (0.4X) resuspended beads to the 100 µl sample from Step 2.3.1. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

2.3.4. Incubate samples for at least 5 minutes at room temperature.

2.3.5. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

2.3.6. After 5 minutes (or when the solution is clear), carefully transfer the supernatant (

140 µl) containing your DNA to a new tube. (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.

0.2X) resuspended SPRIselect or Sample Purification Beads to the supernatant and mix at least 10 times. Be careful to expel all of the liquid from the tip during the last mix. Incubate samples on the bench top for at least 5 minutes at room temperature.

2.3.8. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

2.3.9. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA (Caution: do not discard beads).

2.3.10. Add 200 &mul of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

2.3.11. Repeat Step 2.3.10. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

2.3.12. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

2.3.13. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 &mul 0.1X TE (dilute 1X TE Buffer 1:10 in water).

2.3.14. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

2.3.15. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 &mul to a new PCR tube.

2.3.16. Proceed to PCR Enrichment of Adaptor-ligated DNA in Section 2.4.

Samples can be stored at -20°C.

2.4. PCR Enrichment of Adaptor-ligated DNA

Follow Section 2.4.1A. if you are using the following oligos (10 µM primer): NEBNext Singleplex Oligos for Illumina (NEB #E7350)
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335)
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500)
NEBNext Multiplex Oligos for Illumina (Set 3, NEB #E7710)
NEBNext Multiplex Oligos for Illumina (Set 4, NEB #E7730)
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600)

Follow Section 2.4.1B. if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

2.4.1. Add the following components to a sterile strip tube:

2.4.1A. Forward and Reverse Primers not already combined

Adaptor Ligated DNA Fragments (Step 2.3.16.)
15 µl
(blue) NEBNext Ultra II Q5 Master Mix
25 µl
(blue) Index Primer/i7 Primer*,**
5 µl
(blue) Universal PCR Primer/i5 Primer*, ***
5 µl
Total Volume
50 µl

2.4.1B. Forward and Reverse Primers already combined

Adaptor Ligated DNA Fragments (Step 2.3.16.)
15 µl
/>(blue) NEBNext Ultra II Q5 Master Mix
25 µl
/>(blue) Index/Universal Primer****
10 µl
Total Volume
50 µl

* The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up PCR reactions.

** For use with NEBNext Multiplex Oligos (NEB #E7335, #E7500, #E7710 or #E7730) use only one index primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 primer per reaction.

*** For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.

**** The primers are provided in NEBNext Multiplex Oligos for Illumina (NEB #E6609). Please refer to the NEB #E6609 manual for valid barcode combinations and tips for setting up PCR reactions.

2.4.2. Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

2.4.3. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:

CYCLE STEP
TEMP TIME
CYCLES
Initial Denaturation
98°C 30 seconds
1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 seconds
3-7*
Final Extension
65°C 5 minutes 1
Hold 4°C &infin

* The number of PCR cycles recommended in Table 2.4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 2.4.2 for applications requiring high library yields, such as target enrichment. The number of PCR cycles should be chosen based on input amount and sample type. Thus, samples prepared with a different method prior to library prep may require re-optimization of the number of PCR cycles. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer).

Table 2.4.1


* Cycle number was determined for size selected libraries.
** NEBNext adaptors contain a unique truncated design. Libraries constructed with NEBNext adaptors require a minimum of 3 amplification cycles to add the complete adaptor sequences for downstream processes.


* Cycle number was determined for size selected libraries.

2.4.4. Proceed to Cleanup of PCR reaction in Section 2.5.

2.5. Cleanup of PCR Reaction

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. AMPure XP beads can be used as well. If using AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes before use. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.

2.5.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

2.5.2. Add 45 &mul (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

2.5.3. Incubate samples on bench top for at least 5 minutes at room temperature.

2.5.4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

2.5.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

2.5.6. Add 200 &mul of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

2.5.7. Repeat Step 2.5.6. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

2.5.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

2.5.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 &mul of 0.1X TE (dilute 1X TE Buffer 1:10 in water).

2.5.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

2.5.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 &mul to a new PCR tube and store at &ndash20°C.

2.6. Assess Library Quality on a Bioanalyzer

2.6.1. Dilute library (from Step 2.5.11.) 5-fold in 0.1X TE Buffer.

2.6.2. Run 1 µl on a DNA High Sensitivity Chip.

2.6.3. Check that the library size shows a narrow distribution with an expected peak size based on fragmentation time and size selection (Figure 2.2).

80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer trace, bring up the sample volume (from Step 2.5.11.) to 50 µl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section 2.5.

Figure 2.2: Example of final library size distributions with size selection. Human DNA (NA19240) was fragmented for 5 or 15 minutes.


Protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≤100 ng

1.1.2. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.

Note: It is important to vortex the enzyme mix prior to use for optimal performance.

Figure 1.1: Example of size distribution on a Bioanalyzer ® . Human DNA (NA19240) was fragmented for 5-40 min.

1.1.3. Add the following components to a 0.2 ml thin wall PCR tube on ice:

COMPONENT
VOLUME PER ONE LIBRARY
DNA 26 &mul
(yellow) NEBNext Ultra II FS Reaction Buffer
7 &mul
(yellow) NEBNext Ultra II FS Enzyme Mix
2 &mul
Total Volume
35 &mul

1.1.4. Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.

1.1.5. In a Thermocycler, with the heated lid set to 75°C, run the following program:

5&ndash30 min @ 37°C
30 min @ 65°C
Hold @ 4°C

If necessary, samples can be stored at &ndash20°C however, a slight loss in yield (

20%) may be observed. We recommend continuing with adaptor ligation before stopping.

Determine whether adaptor dilution is necessary.

If DNA input is < 100 ng, dilute the (red) NEBNext Adaptor for Illumina in 10 mM Tris-HCl, pH 7.5-8.0 with 10 mM NaCl as indicated in Table 1.2.1.

Table 1.2.1: Adaptor Dilution

INPUT
ADAPTOR DILUTION
(VOLUME OF ADAPTOR:TOTAL
VOLUME)
WORKING ADAPTOR
CONCENTRATION
100 ng-500 ng
No Dilution
15 µM
5 ng-99 ng
10-fold (1:10)
1.5 µM
less than 5 ng
25-fold (1:25)
0.6 µM

Note: The appropriate adaptor dilution for your sample input and type may need to be optimized experimentally. The dilutions provided here are a general starting point.

1.2.1. Add the following components directly to the FS Reaction Mixture:

COMPONENT VOLUME
FS Reaction Mixture (Step 1.1.5)
35 µl
(red) NEBNext Ultra II Ligation Master Mix*
30 µl
(red) NEBNext Ligation Enhancer
1 µl
(red) NEBNext Adaptor for Illumina**
2.5 µl
Total Volume
68.5 µl

* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
** The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535 and #E6609) Oligos for Illumina.

Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend adding adaptor to a premix in the Adaptor Ligation Step.

1.2.2. Set a 100 µl or 200 µl pipette to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. (Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).

1.2.3. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid off.

1.2.4. Add 3 µl of (red) USER ® Enzyme to the ligation mixture from Step 1.2.3.

Note: Steps 1.2.4. and 1.2.5. are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535 and #E6609) Oligos for Illumina.

1.2.5. Mix well and incubate at 37°C for 15 minutes with the heated lid set to &ge 47°C.

Samples can be stored overnight at -20°C.

1.3. Size Selection or Cleanup of Adaptor-ligated DNA

The following section is for cleanup of the ligation reaction for inputs &le 100 ng. If your input DNA is > 100 ng, follow the size selection protocol in Chapter 2, Section 2.3. If you want fragment sizes > 550 bp and your input is &ge 100 ng, follow the entire protocol in Chapter 3.

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step (71.5 µl Step 1.2.5.). AMPure XP Beads can be used as well. If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use. These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.

1.3.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

1.3.2. Add 57 &mul (0.8X) resuspended beads to the Adaptor Ligation reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

1.3.3. Incubate samples for at least 5 minutes at room temperature.

1.3.4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

1.3.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

1.3.6. Add 200 &mul of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

1.3.7. Repeat Step 1.3.6. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

1.3.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

1.3.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 &mul 0.1X TE (dilute 1X TE Buffer 1:10 in water).

1.3.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

1.3.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 &mul to a new PCR tube.

1.3.12. Proceed to PCR Enrichment of Adaptor-ligated DNA in Section 1.4.

Samples can be stored overnight at -20°C.

1.4. PCR Enrichment of Adaptor-ligated DNA

Follow Section 1.4.1A. if you are using the following oligos (10 µM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350)
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335)
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500)
NEBNext Multiplex Oligos for Illumina (Set 3, NEB #E7710)
NEBNext Multiplex Oligos for Illumina (Set 4, NEB #E7730)
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600)

Follow Section 1.4.1B. if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

1.4.1. Add the following components to a sterile strip tube:

1.4.1A. Forward and Reverse Primers not already combined

Adaptor Ligated DNA Fragments (Step 1.3.12.)
15 µl
(blue) NEBNext Ultra II Q5 Master Mix
25 µl
(blue) Index Primer/i7 Primer*,**
5 µl
(blue) Universal PCR Primer/i5 Primer*, ***
5 µl
Total Volume
50 µl

1.4.1B. Forward and Reverse Primers already combined

Adaptor Ligated DNA Fragments (Step 1.3.12.)
15 µl
/>(blue) NEBNext Ultra II Q5 Master Mix
25 µl
/>(blue) Index/Universal Primer****
10 µl
Total Volume
50 µl

* The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up PCR reactions.

** For use with NEBNext Multiplex Oligos (NEB #E7335, #E7500, #E7710 or #E7730) use only one index primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 primer per reaction.

*** For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.

**** The primers are provided in NEBNext Multiplex Oligos for Illumina (NEB #E6609). Please refer to the NEB #E6609 manual for valid barcode combinations and tips for setting up PCR reactions.

1.4.2. Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

1.4.3. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:

CYCLE STEP
TEMP TIME
CYCLES
Initial Denaturation
98°C 30 seconds
1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 seconds
3-13*
Final Extension
65°C 5 minutes 1
Hold 4°C &infin

* The number of PCR cycles recommended in Table 1.4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 1.4.2 for applications requiring high library yields, such as target enrichment. The number of PCR cycles should be chosen based on input amount and sample type. Thus, samples prepared with a different method prior to library prep may require re-optimization of the number of PCR cycles. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer).

1.4.4. Proceed to Cleanup of PCR reaction in Section 1.5.

1.5. Cleanup of PCR Reaction

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. AMPure XP beads can be used as well. If using AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes before use. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.

1.5.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

1.5.2. Add 45 &mul (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

1.5.3. Incubate samples on bench top for at least 5 minutes at room temperature.

1.5.4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

1.5.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

1.5.6. Add 200 &mul of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

1.5.7. Repeat Step 1.5.6. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

1.5.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

1.5.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 &mul of 0.1X TE (dilute 1X TE Buffer 1:10 in water).

1.5.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

1.5.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 &mul to a new PCR tube and store at &ndash20°C.

1.6. Assess Library Quality on a Bioanalyzer

1.6.1. Dilute library (from Step 1.5.11.) 5-fold in 0.1X TE Buffer (inputs &le 1 ng may not require dilution to run on a Bioanalyzer).

1.6.2. Run 1 µl on a DNA High Sensitivity Chip.

1.6.3. Check that the library size shows a narrow distribution with an expected peak size based on fragmentation time (Figure 1.2).

80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer trace, bring up the sample volume (from Step 1.5.11.) to 50 µl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section 1.5. You may see adaptor-dimer when starting with inputs &le 1 ng

Figure 1.2: Example of final library size distributions without size selection. Human DNA (NA19240) was fragmented for 5-40 minutes.


Author information

These authors contributed equally: J. Blackburn, T. Wong.

Affiliations

Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, Australia

James Blackburn, Ted Wong, Bindu Swapna Madala, Chris Barker, Simon A. Hardwick, Andre L. M. Reis, Ira W. Deveson & Tim R. Mercer

St Vincent’s Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia

James Blackburn, Simon A. Hardwick, Andre L. M. Reis, Ira W. Deveson & Tim R. Mercer

Altius Institute for Biomedical Sciences, Seattle, WA, USA

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

Contributions

J.B., B.S.K. and C.B. contributed materials. J.B. performed the experiments. T.W., I.W.D., S.A.H. and A.L.M.R. carried out the bioinformatic analysis. J.B., T.W., I.W.D. and T.R.M. wrote the manuscript. All authors conceived the study and contributed to manuscript preparation.

Corresponding authors


How to make a DNA ladder? a DIY guide

The ligation-based method is one of the traditional methods not used nowadays.

In the ligation-based DNA ladder development, different fragments of 100bp are covalently joined by the phosphodiester bonds that generated different fragments of 200bp to 1000bp.

The graphical representation of Ligation based DNA ladder development method.

In the restriction digestion method, the know restriction endonuclease is used to digest different fragments of DNA that generate different-sized fragments of DNA.

The digested fragments are collected, purified and can be used as a DNA ladder.

However, both the methods are outdated, costlier and time-consuming. In addition to this, the amount of fragment generated from both methods is very low.

Therefore, the need for the new method has arisen which may be cheap, rapid and most importantly generates a large amount of specific DNA fragments for the construction of DNA ladder.

We know that only PCR can generate millions of fragments in a short time.

PCR is used to generate different types of DNA fragments for the construction of a DNA ladder.

In the very first step, we have to select the plasmid. Use bacteria phage plasmid.

Now digest the plasmid with the appropriate restriction endonuclease so that the circular DNA breaks open.

Here we have selected lambda phage DNA sequence between the sequence 6631 to 7630 and designed primers according to it.

We have designed a set of primers that can amplify different fragments of DNA in a multiplex reaction.

For achieving amplification for all the fragments, we need an advance PCR reaction preparation set up.

For instance, the R1 primer is used in all the amplification, therefore we have to add it 10 times more than other primers.

You can also read our PCR reaction preparation guide to learn how to prepare an effective PCR reaction: PCR reaction: Ten secrets that nobody tells you

The present method is a combination of three different PCR methods: Hot start PCR, multiplex PCR and touch down PCR.

Amplification of all the fragments are carried out in a single reaction (multiplex), Temperature gradually decreases to increase the amplification power of PCR (touch down) and the Taq DNA polymerase used in the present experiment is added only when the reaction is started (hot start).

Note: The method is a touchdown PCR, therefore, decrease the temperature after 2 PCR cycles until the temperature reaches 44 ̊C.

After the completion of the PCR reaction, the products are loaded on the 2% agarose gel.

Want to learn how to prepare an agarose gel? read this article: Agarose gel electrophoresis

Run the gel on 80V until the DNA reaches up to the 75% distance of the gel.

10 different DNA bands are obtained from the PCR reaction and are compared with the ready to use DNA ladder.

Now, our DNA ladder is ready to use, but before that, we have to purify the fragments.

For that precipitate the fragments using the alcohol and purify it with the ready to use DNA purification kit.

You can also use the phenol-chloroform method for DNA purification (Here I prefer to use a kit based method over the PCI method).

Again dissolve the fragments in TE buffer and store it under the cooling conditions.

Now you can use 5μl of our DNA ladder for any of the gel electrophoresis experiment.

If you are not comfortable with multiplexing the reaction (because more extraordinary expertise required to achieve good results in multiplex PCR),

Then perform the PCR reaction in 10 different tubes and load it on agarose gel along with the ready to use DNA ladder for conforming results.

The results of the PCR doing in 10 different separate reactions might look like this,


Acknowledgements

D.D.D.C. and S.V.B. were supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre. J.M.B. was supported by a fellowship from the Strategic Training in Transdisciplinary Radiation Science for the 21st Century (STARS21) training program. D.D.D.C. was supported by the University of Toronto McLaughlin Centre (MC-2015-02) the Canadian Institutes of Health Research (CIHR FDN 148430 and CIHR New Investigator Salary award 201512MSH-360794-228629) the Ontario Institute for Cancer Research (OICR), with funds from the province of Ontario the Canada Research Chair (950-231346) and the Helen M Cooke Professorship from the Princess Margaret Cancer Foundation. S.V.B. was supported by a Career Development Award from the Conquer Cancer Foundation of ASCO. Any opinions, findings, and conclusions expressed in this article are those of the author(s) and do not necessarily reflect those of the American Society of Clinical Oncology or the Conquer Cancer Foundation. We acknowledge the Princess Margaret Cancer Centre Head & Neck Translational Program, supported by philanthropic funds from the Wharton Family, Joe’s Team, Gordon Tozer and the Reed Fund, as well as the labs of F.-F. Liu and G. Liu (University of Toronto), for the provision of plasma samples. We also thank the Princess Margaret Genomics Centre for carrying out the NGS sequencing and the Bioinformatics and HPC Core of the Princess Margaret Cancer Centre for expertise in generating the NGS data.


Access options

Get full journal access for 1 year

All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.

Get time limited or full article access on ReadCube.

All prices are NET prices.


Stroke: Injury Mechanisms

A.B. Singhal , . S.P. Finklestein , in Encyclopedia of Neuroscience , 2009

Apoptosis

Apoptosis is an evolutionarily conserved process of programmed cell death. It is characterized histologically by nuclear and cytoplasmic condensation, nuclear fragmentation, and cell shrinkage. Apoptotic cells are terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells that exhibit DNA laddering . Cell type, age, and brain location render them more or less resistant to apoptosis or necrosis. Since apoptotic pathways require energy in the form of ATP, apoptosis predominantly occurs in the ischemic penumbra that sustains milder injury, rather than in the core, where ATP levels are rapidly depleted. Nevertheless, ionic imbalances can also trigger apoptosis under certain conditions, suggesting a close mechanistic interrelationship between glutamate-mediated excitotoxicity and apoptosis. The normal human brain expresses caspases 1, 3, 8, and 9, apoptosis protease-activating factor-1 (APAF-1), death receptors, P53, and a number of Bcl-2 family members, all of which are implicated in apoptosis.

Caspases are protein-cleaving enzymes (zymogens) that belong to a family of cysteine aspartases constitutively expressed in adult and newborn brain cells, particularly neurons. Apoptosis occurs via caspase-dependent as well as caspase-independent mechanisms. Caspases are sequentially cleaved and activated via two pathways: an extrinsic pathway dependent on death receptors, and an intrinsic mitochondrial pathway. With regard to the extrinsic pathway, several apoptotic triggers of have been identified. These include oxygen free radicals, Bid cleavage, death receptor ligation, DNA damage, and lysosomal protease activation. The tumor necrosis factor (TNF) superfamily of death receptors, most importantly Fas, powerfully regulates upstream caspase processes. Emerging data suggest that the cell nucleus is involved in releasing signals for apoptosis, and that the mitochondrion plays a central role in mediating apoptosis. At least four mitochondrial molecules mediate downstream cell death pathways: cytochrome c, secondary mitochondria-derived activator of caspase (Smac/Diablo), apoptosis-inducing factor (AIF), and endonuclease G. While cytochrome c and Smac/Diablo mediate caspase-dependent apoptosis, AIF and endonuclease G mediate caspase-independent apoptosis. Cytochrome c binds to Apaf-1, which combines with procaspase 9 to form the ‘apoptosome’ that activates caspase 9 in the presence of dATP. In turn, caspase 9 activates caspase 3. It is important to note that formation of the apoptosome complex is inhibited by Bcl-2, which becomes upregulated very early after stroke. Smac/Diablo binds to inhibitors of activated caspases and causes further caspase activation. After activation, caspases 3 and 7 (the executioner caspases) attack and degrade various substrate proteins, ultimately leading to DNA fragmentation and irreversible cell death. Over 30 proteins can be cleaved, including the DNA-repairing enzyme poly(ADP-ribose) polymerase (PARP), the cytoskeletal protein gelsolin, actin, presenilins, and other caspases. Caspase 3, the most prominent cysteine protease, is present in the infarct core as well as in the penumbra and is cleaved acutely after stroke. Subsequent caspase cleavage occurring after hours or days leads to delayed cell death.

Caspase-independent apoptosis has been shown to develop in cultured neurons by NMDA-receptor induced activation of PARP-1, and in fibroblasts exposed to oxidative stress. AIF is implicated as a key molecule in this cascade. AIF is released from mitochondria after PARP-1 activation, then relocates to the nucleus, where it binds DNA, promotes chromatin condensation, and leads to cell death via incompletely understood mechanisms. The exact significance of caspase-independent apoptosis remains to be determined.

Overexpression of Bcl-2, deletions of genes for Bid or caspase-3, and the use of caspase-3 inhibitors, peptide inhibitors, and antisense oligonucleotides that suppress the expression and activity of apoptosis-promoting genes have been shown to reduce infarct volume or decrease neurological deficits after stroke. A single intracerebroventricular injection of zDEVD-FMK, a relatively selective caspase-3 inhibitor, is shown to be neuroprotective when administered up to 9 h after transient focal stroke. However, caspase inhibitors do not reduce infarct size in all brain ischemia models. This variable effect may be attributable to different severities of ischemia, to limited potency or inability of caspase inhibitors to cross the blood–brain barrier, to the relatively minor impact of apoptosis as compared to excitotoxicity or other mechanisms on stroke outcome, and to upregulation of caspase-independent or redundant cell death pathways.


Watch the video: How to Load an Agarose Gel (November 2022).