Dinh/Dinh 2015/NOTES/2015-3-21: Difference between revisions

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|-
|-
| oxBS-F/-||8.11||5||40.55||0||2.5||0||0||17.5
| oxBS-F/-||8.11||5||40.55||0||2.5||0||0||17.5
|}
* Mix gently
* Incubate the repair reaction for 20 minutes at 37C.
* Place the reactions on ice
* Proceed with PCR amplification
* Reaction: make 3 different master mixes for B,E,F primers
{| class="wikitable"
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Concentration'''
| align="center" style="background:#f0f0f0;"|'''1X volume'''
| align="center" style="background:#f0f0f0;"|'''Final concentration'''
| align="center" style="background:#f0f0f0;"|'''4.2X volume'''
|-
| Nuclease free water||NA||34.45||NA||144.69
|-
| Phusion HF Buffer||10X||10||1X||42
|-
| Primer F/R (B/E/F)||10 uM||1.5||300 nM||6.3
|-
| dNTP||10 mM||1.25||250 nM||5.25
|-
| Phusion U||2U/uL||0.8||1.6 U||3.36
|-
| Template||NA||2||NA||NA
|}
|}

Revision as of 19:02, 31 March 2015

Control DNA Design v3

  • This week I began working on a new design for control DNA, hopefully I will only spend 1 extra week on this design.
  • I realized that I dont have to continue using the Digestion Control DNA sequence in the TruMethyl kit. I can design my control DNA from Lambda Phage DNA sequences. I used BiSearch online software to look for sequences that are suitable for amplification post-bisulfite covnersion with high specificity. I picked the top ranked result from BiSearch, extracted the unconverted sequence, and checked NEB Cutter for potential digestion site. I found that the restriction enzyme SphI cuts this 277 bp fragment at GCATGC recognition site. This enzyme is not methylation sensitive and so should be good to use for digestion-qPCR test of conversion efficiency. Since MspI appears to have some methylation sensitivity, I decided to not use this enzyme.
  • There are a total of 6 SphI digestion sites on Lambda Phage DNA, and I designed PCR primers for converted and unconverted DNA to generate 160-300 bp fragments with SphI digestion site in the middle. The primers are named A, B, C, D, E, and F.

Test A-F Lambda Phage DNA primers

  • For bisulfite converted DNA, we converted Lambda Phage DNA (NEB) using the EZ-Methylation Lightnigh Kit. The converted DNA was diluted to ~3ng/uL
  • Reaction for bisulfite converted DNA:
 Primers F/R mix (5 uM)                2 uL
 Template (~3ng/uL)                    1 uL
 2x iQ master mix                      25 uL
 Nuclease free water                   22 uL
  • Reaction for non-converted DNA:
 Primers F/R mix (5 uM)                2 uL
 Template (~1ng/uL)                    1 uL
 2x Taq master mix                     25 uL
 Nuclease free water                   22 uL
  • Program (same for both converted and non-converted reactions):
 96C 3 min -> [95C 30s, 55C 30s, 72C 30s]x20cycles -> 72C 1 min -> 4C hold
  • Gel image:

File:Control dna v3 unconverted lambdaDNA pcr.jpg File:Control dna v3 converted lambdaDNA pcr.jpg

  • Pooled B, E, F amplicons for size selection. Want to have a little background as possible for amplification.

PCR control DNA with modifications

  • Prepare 800 uL reaction for each. Use primers B for 5hmC, primers E for 5mC, and primers F for C.
  • Reaction set up:
 10x PCR buffer                10 uL x 8 = 80 uL
 water                         72 uL x 8 = 576 uL
 MgCl2                          7 uL x 8 = 56 uL
 Primer F/R (5uM)               4 uL x 8 = 32 uL
 Pooled B,E,F amplicons         1 uL x 8 = 8 uL
 JumpStart Taq Pol              2 uL x 8 = 16 uL
 dNTP mix                       4 uL x 8 = 32 uL
  • Program:
 94C 1 min -> [94C 30s, 55C 30s, 72C 1 min] x 20 cycles -> 72C 1 min -> 4C hold
  • Purified amplicons with QIAX II beads
  • Nanodrop
 B (5hmC) = 57.6 ng/uL
 E ( 5mC) = 85.4 ng/uL
 F (C)    = 57.4 ng/uL

T4 PNK + T4 DNA Ligase + TruSeq Adaptor Ligation

  • Preparing adapted controls for testing only!!!
  • Pool 1600 ng of each control DNA for testing, split into 9 reactions with ~500 ng pooled control amplicons in each.
  • Reaction set up:
 T4 PNK (10U/uL)             1 uL
 10x T4 DNA ligase buffer    1 uL
 Pooled amplicons (~500ng)   8 uL
  • 37C for 40 minutes
  • Add ligation reagents:
 10x T4 DNA ligase buffer    1 uL
 T4 DNA Ligase (6U/uL)       1 uL
 TruSeq Adaptors             1 uL
 Nuclease-free water         7 uL
  • 16C overnight. (18 hours)
  • Purified with 40 uL AMPure beads.
    • To prepare for the oxidation, we ran the entire volume of purified ligation products through a BioRad P6 SSC column, but because the column had leftover water in it, we diluted the ligation products to >500 uL volumes. We had to concentrate them again using QIAX II beads.
  • Tube ID & adaptor used:
 1 - 13
 2 - 14
 3 - 15
 4 - 16
 5 - 27
 6 - 18
 7 - 19
 8 - 20
 9 - No adaptor added.

PAGE Gel after qPCR with Illumina primers

  • Control B (5hmC) is 189 bp + 120 bp = 309 bp
  • Control E (5mC) is 262 bp + 120 bp = 382 bp
  • Control F (C) is 277 + 120 bp = 397 bp
  • File:Control dna v3 adapted qPCR.jpg
  • Reaction #9 with no adaptor added did not amplify.
  • qPCR concentrations
Tube ID ng/uL
9 0
1 9.367248
2 9.665568
3 11.3162709
4 10.29204
5 9.128592
6 10.4014251
7 9.2678091
8 10.5406389

oxBS Test 1

  • Testing these 8 conditions:
 (1) 1 ug Lambda DNA + 50 ng adapted control DNA + 15 nmoles oxidant (more bisulfite)
 (2) 1 ug Lamdba DNA + 50 ng adapted control DNA + 22.5 nmoles oxidant (more bisulfite)
 (3) 1 ug Lambda DNA + 50 ng adapted control DNA + 30 nmoles oxidant (more bisulfite)
 (4) 500 ng Lambda DNA + 25 ng adapted control DNA + 15 nmoles oxidant (more bisulfite)
 (5) 500 ng Lamdba DNA + 25 ng adapted control DNA + 22.5 nmoles oxidant (more bisulfite)
 (6) 500 ng Lambda DNA + 25 ng adapted control DNA + 30 nmoles oxidant (more bisulfite)
 (7) 1 ug Lambda DNA + 50 ng adapted control DNA + 15 nmoles oxidant (less bisulfite)
 (8) 500 ng Lambda DNA + 25 ng adapted control DNA + 15 nmoles oxidant (less bisulfite)
More bisulfite: use twice as much bisulfite mix per oxidation reaction
Less bisulfite: use normal amount of bisulfite mix per oxidation reaction
  • Prepare each mixure condition types using final volume of 23 uL, each condition uses control DNA with a different Indexed adaptor for sequencing.
  • Use BioRad P6 SSC columns to buffer exchange each 23 uL mix.
  • Prepare fresh 15 M NaOH, dilute to 1 M NaOH. Denature each mix with 1.25 uL of 1 M NaOH for 30 minutes.
  • Prepare fresh 2.155 M KRuO4: 0.044 g KRuO4 + 56 uL water, dilute to 15 mM, 22.5 mM, and 30 mM using fresh 1 M NaOH and water.
 0.70 uL KRuO4 (2.155M) + 5 uL NaOH (1M) + 94.30 uL water
 1.05 uL KRuO4 (2.155M) + 5 uL NaOH (1M) + 93.95 uL water
 1.40 uL KRuO4 (2.155M) + 5 uL NaOH (1M) + 93.60 uL water
  • Add 1 uL of KRuO4 solution (corresponding concentration) to each test condition. Oxidize at 40C for 1 hour.
  • After incubation the reactions were all clear with maybe a slight orange tinge.
  • Bisulfite conversion reaction set up:
 More bisulfite: To each oxidation reaction add 15 uL water (to 40 uL), 170 uL dissolved Bisulfite Mix,70 uL of DNA Protect Buffer.
 Less bisulfite: To each oxidation reaction add 85 uL dissolved Bisulfite Mix, 35 uL of DNA Protect Buffer.
  • Reaction program (~ 8hrs):
 95C 5 min -> 60C 20 min
 95C 5 min -> 60C 40 min
 95C 5 min -> 60C for 165 min
 95C 5 min -> 60C 20 min
 95C 5 min -> 60C 40 min
 95C 5 min -> 60C for 165 min
 20C hold overnight.

Post oxBS quantification (Nanodrop)

 1 - 72.6 ng/uL
 2 - 85.2 ng/uL
 3 - 154.3 ng/uL
 4 - 53.4 ng/uL
 5 - 97.8 ng/uL
 6 - 68.0 ng/uL
 7 - 38.0 ng/uL
 8 - 45.4 ng/uL

Post oxBS PCR of control DNA with TruS_F/R primers

  • Reaction set up:
Reagent Concentration Volume (uL) Final concentration 8.5X volume
Water NA 40.65 NA 345.53
PfuTurboCx Buffer 10X 5 1X 42.5
TruS_F 100 uM 0.15 0.3 uM 1.275
TruS_R 100 uM 0.15 0.3 uM 1.275
dNTP 10 mM 1.25 250 nM 10.625
PfuTurboCx 2.5U/uL 0.8 1 U 6.8
Template NA 2 NA ---
  • I didn't get the 5hmC control out from the PCR. This is a big problem because without the 5hmC control we cannot determine the effectiveness of oxidation. I only saw bands at ~400 bp which means the 5mC and/or C survived the whole process.
  • 2 uL template (post oxBS elution), 20 cycles:

File:Control dna v3 post oxBS pcr 2ul.jpg

  • 10 uL template (post oxBS elution), 15 cycles:

File:Control dna v3 post oxBS pcr 10ul.jpg

qPCR, quantify how much lambda DNA leftover from conversion

    • First I purified amplified converted lambda DNA using primer set D and quantified the first dilution using Qubit. Got 2.48 ng/uL
    • Next, I made serial dilution by 10 folds less each time, using 10 mM Tris-HCl (pH 8.5), .5% Tween 20 solution
    • For each sample, I diluted 400X (or 1000X for sample 1), using 199.5 uL 10 mM Tris-HCl (pH 8.5), .5% Tween 20 solution
    • Made a master mix then add master mix to triplicates of Standards and samples.
Reagent Starting concentration Volume (uL) Final concentration 50X
iQ Supermix Green 2X 5 1X 250
Primer F/R 10 uM 0.3 300 nM 15
Template NA 4.7 NA NA
  • Results from iQ looked ok but after running PAGE gel, amplification were of backgrounds or primer dimers. I need to repeat this using more diluted Standards as well.
  • qPCR, repeat with Phusion U
Reagent Starting concentration Volume (uL) Final concentration 50X
Nuclease-free water NA 4.48 NA 224
Sybr Green 50X 0.32 0.8X 16
Phusion HF Buffer 5X 4 1X 200
dNTPs 10mM 0.4 200 nM 20
Primer F/R 10uM 0.6 300 nM 30
Phusion U HotStart Pol 2 U/uL 0.2 0.02 U/uL 10
Template 10 NA ---

File:Control dna v3 qPCR post-oxbs lambdaD std.jpg File:Control dna v3 qPCR post-oxbs lambdaD.jpg

  • Concentration:
    • Normalize concentration: sybr(ng/uL) * 48502 bp/267 bp (Std D) * 400 (dilution factor)
    • Dilution factor for #1 is 1000, only used 0.2 uL in 200 uL
1. 32.3 ng/uL; 646 ng
2. 19.7 ng/uL; 394 ng
3. 59.4 ng/uL; 1190 ng
4. 8.14 ng/uL; 163 ng
5. NA
6. 8.42 ng/uL; 168 ng
7. 11.1 ng/uL; 223 ng
8. 7.99 ng/uL; 160 ng

Conclusion from oxBS test

  • Still waiting on sequencing to determine the conversion rates between 2x bisulfite mix versus 1x bisulfite mix per reaction. The yield from using 2x bisulfite mix was much less for the 1 ug case and about the same for the 500 ng case.
  • On increasing the concentration of oxidant, for the 1 ug case, in order of increasing oxidant we got: 646 ng -> 394 ng -> 1190 ng. In the last case, we might have errored on pipetting, so considering a 50% reduction in yield, we might need to consider using the smaller concentration of oxidant. For the 500 ng case, we only got data for the 1st and 3rd levels of oxidant concentrations, the yield were 163 ng -> 168 ng. Again, we are not sure why the 3rd level of oxidant always yielded the higher amount of DNA, and this may be due to pipetting error. I will repeat the experiment to compare the 2nd and 3rd level of oxidant.
  • We added 5% w/w control DNA but was not able to get the 5hmC back out. We probably need to increase the percentage of control DNA added, considering that up to over 80% loss of DNA was observed. Furthermore, not all of the control DNA may have adaptors on them since we cannot isolate those out without performing PCR. We cannot perform PCR on the adapted control DNA because we need the methylated adaptors on them.
  • Since the yield from 500 ng of input DNA is >100 ng each time, we can continue with capture with a starting amount of 500 ng for oxBS.

oxBS Test 2

  • Testing these 4 conditions:
 (5) 500 ng Lambda DNA + 100 ng adapted control DNA + 15 nmoles oxidant (more bisulfite)
 (6) 500 ng Lamdba DNA + 100 ng adapted control DNA + 22.5 nmoles oxidant (more bisulfite)
 (7) 500 ng Lambda DNA + 100 ng adapted control DNA + 30 nmoles oxidant (more bisulfite)
 (8) 500 ng Lambda DNA + 100 ng adapted control DNA + 30 nmoles oxidant (less bisulfite - used all leftover bisulfite mix)
  • Added 1.5 uL Lambda DNA + 6 uL adapted control DNA (~10 ng/uL) + 15.5 uL oxidant
  • Buffer exchange with P6 columns
  • Added 20 uL buffer exchanged DNA + 1.1 uL 1M NaOH, incubate for ~40 min at 37C
  • Added 1 uL of KRuO4 mix (concentrations: 15 mM, 22.5 mM, and 30 mM), incubate for 1 hr at 40C
  • Added 170 uL bisulfite mix, 70 uL DNA protect buffer, and 18 uL water, start bisulfite conversion program (~8 hr).
  • After bisulfite clean-up, perform PCR on 2 uL of oxBS products
  • Reaction
Reagent Concentration 1X volume Final concentration 4.5X volume
Nuclease-free water NA 35.65 NA 160.425
Phusion HF Buffer 5X 10 1X 45
TruSeq F/R primer 10 uM 1.5 300 nM 6.75
dNTP 10 mM 1.25 250 nM 5.625
Phusion U 2 U/uL 0.8 1.6 U 3.6
Template NA 2 NA ---
  • Gel
File:Control dna v3 test2 post oxBS pcr.jpg

oxBS test - 5hmC control DNA damage/fragmentation?

  • Puzzled why 5hmC DNA control were always missing or unable to amplify with PCR after oxBS.
  • Repeat experiment with unadapted control DNA: if extensive DNA fragmentation is occurring we should be smears instead of distinct bands for the modified amplicons.
  • Amplicons:
 B (5hmC) - 189 bp
 E (5mC) - 262 bp
 F (C) - 277 bp
  • First prepare BioRad P6 SSC columns and add DNA diluted with water to 25 uL volume.
  • Dilute 15 M NaOH to 1 M NaOH, added 1.25 uL to each and incubate at 37C for 30 minutes to denature
  • For oxidation, dilute KRuO4 stock to 15 mM with water and 1 M NaOH.
  • After oxidation, ran a PAGE gel to check if oxidation fragmented the DNA.

File:Control dna v3 test oxidation fragmentation.jpg

  • Next, since we were out of the EpiTect Bisulfite Conversion reagent, we used Zymo's EZ Methylation Lightning Kit.
  • Added 130 uL conversion reagent to 20 uL oxidized DNA, for control, use 500 ng of unoxidized DNA.
  • After bisulfite conversion cleanup, ran a denaturing PAGE gel to check if oxBS fragmented the DNA:

File:Control dna v3 test oxBS fragmentation.jpg

  • Qubit ssDNA :
 1) bs - B - 39.4 ng/uL (551 ng)
 2) bs - E - 26.6 ng/uL (372 ng)
 3) bs - F - 12.76 ng/uL (178 ng)
 4) oxBS - B - 10.54 ng/uL (147 ng)
 5) oxBS - E - 2.86 ng/uL (40 ng)
 6) oxBS - F - 8.11 ng/uL (113 ng)
  • oxBS yielded much less DNA than BS alone thus DNA most likely were fragmented. However, we see this for all of the modified DNA, not just B (5hmC), so this doesn't explain why 5hmC control were always missing after oxBS.
  • Next, we needed to test whether the oxBS reduced the ability of the DNA to polymerize due to oxidative damage. We will use NEB's PreCR Repair Mix to treat half of the DNA, and then try to amplify the treated and untreated DNA with PCR to see whether one will amplify better than the other.

PreCR Repair Mix

  • Reaction:
Sample Concentration(ng/uL) DNA volume Mass (ng) 10 mM dNTPs 10X ThermoPol Buffer 100X NAD+ PreCR Repair Mix Water
bs-B 39.4 5 197 0.25 2.5 0.25 0.5 16.5
bs-E 26.6 5 133 0.25 2.5 0.25 0.5 16.5
bs-F 12.76 5 63.8 0.25 2.5 0.25 0.5 16.5
oxBS-B 10.54 5 52.7 0.25 2.5 0.25 0.5 16.5
oxBS-E 2.86 5 14.3 0.25 2.5 0.25 0.5 16.5
oxBS-F 8.11 5 40.55 0.25 2.5 0.25 0.5 16.5
bs-B/- 39.4 5 197 0 2.5 0 0 17.5
bs-E/- 26.6 5 133 0 2.5 0 0 17.5
bs-F/- 12.76 5 63.8 0 2.5 0 0 17.5
oxBS-B/- 10.54 5 52.7 0 2.5 0 0 17.5
oxBS-E/- 2.86 5 14.3 0 2.5 0 0 17.5
oxBS-F/- 8.11 5 40.55 0 2.5 0 0 17.5
  • Mix gently
  • Incubate the repair reaction for 20 minutes at 37C.
  • Place the reactions on ice
  • Proceed with PCR amplification
  • Reaction: make 3 different master mixes for B,E,F primers
Reagent Concentration 1X volume Final concentration 4.2X volume
Nuclease free water NA 34.45 NA 144.69
Phusion HF Buffer 10X 10 1X 42
Primer F/R (B/E/F) 10 uM 1.5 300 nM 6.3
dNTP 10 mM 1.25 250 nM 5.25
Phusion U 2U/uL 0.8 1.6 U 3.36
Template NA 2 NA NA