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

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* Results from Phusion U
* Results from Phusion U
** After qPCR I ran a gel to confirm that the correct size products were generated. Expected 267 bp band for LambdaD DNA.
** After qPCR I ran a gel to confirm that the correct size products were generated. Expected 267 bp band for LambdaD amplicons.
** [[File:control_dna_v3_qPCR_lambdaD_std_gel.jpg | 300px]] [[File:control_dna_v3_qPCR_lambdaD_post_oxBS_gel.jpg | 300px]]  
** [[File:control_dna_v3_qPCR_lambdaD_std_gel.jpg | 300px]] [[File:control_dna_v3_qPCR_lambdaD_post_oxBS_gel.jpg | 300px]]  
[[File:control_dna_v3_qPCR_post-oxbs_lambdaD_std.jpg | 500px]]
[[File:control_dna_v3_qPCR_post-oxbs_lambdaD_std.jpg | 500px]]

Revision as of 17:15, 25 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

  • 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.
  • 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 bisulfite 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 bisulfite conversion 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