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 | ** 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 | --- |
- 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 amplicons.
- File:Control dna v3 qPCR lambdaD std gel.jpg File:Control dna v3 qPCR lambdaD post oxBS gel.jpg
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