Noi/NOTES/2013-5-30
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DNA methylation assay for Blueprint project
2013-05-30
- Received oligos from CustomArray
- Oligo information
- Design: BSPP_Design-5-17-2013 --> I would name BSPP_Blueprint for future experiment
- Length 150 mer (MW = 150 * 330Da/bp = 49,500 Da or g/mole
- Cocn. 40.16ng/ul = 811.3 nM (40.16ng/ul/ 49,500 g/mole)
- Volume 80ul in TE buffer. Total amount 3.21ug.
- Probe prep plan
- Last time of Matt's experiment on Expansion PCR of oligo from CustomArray, we need to used template ~100x more than probe synthesized from Agilent. This time I will test for expansion PCR using different concentration of oligo template (concentration in expansion PCR 20nM and 100nM) and use DMR330k oligos (Exp1) from Agilent as PC.
- Aliquot 40ul oligo from original tube and dilute to 500nM (mix 40ul of 811.3nM oligo with 24.90ul H2O)
Expansion PCR (TEST)
Components | Volume (ul) | Final conc. | Volume for 3.5 rxn mix |
500nM seed oligoes | 2.00 or 5.00 | 20nM or 50nM | 0.00 |
AP1V6 (100uM) | 0.20 | 400nM | 0.70 |
AP2V6 (100uM) | 0.20 | 400nM | 0.70 |
2x KAPA SYBG fast MM | 25.00 | 1x | 87.50 |
H2O | 19.60 | 68.60 | |
Total | 45.00 | 157.50 |
- Aliquot 45ul, add oligo template, and adjust total volume to 50ul with H2O
- Add 2.5 ul of DMR330k Exp1, final conc. in expansion PCR is 1nM
Program 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold
File:20130530 BSPPBlueprint TESTExpensionPCR.png
- from qPCR curve, seemed like 20nM and 50nM didn't amplify very well comparing to PC as expected (similar to Matt's result using the oligos from the same source), so after PAGE verification, I will do expansion PCR with 100nM template.
PAGE analysis to verify amplified amplicons in 6% TBU gel
- Loaded 2.5ul of PCR products in TBU gel
- I loaded amplified amplicons in TBU gel becuase I dodn't want to waste the TBU gel used for single strand seed oligos verification
File:ZhangLab 2 2013-05-30 15hr 54min TESTExpansionPCR.jpg Lane1: 10bp DNA ladder Lane2: O = 3ul of 500nM oligo before expansion PCR Lane3: PC (BSPP DMR330k Exp1) Lane4: 20nM BSPP _Blueprint after expansion PCR Lane5: 50nM BSPP _Blueprint after expansion PCR
- From the gel image above, the expected band of ~150 bp was amplified.
- The seed oligos from original tube before expansion PCR show a smear as observed in Matt' oligos.
- There was the extra band in PC of DMR330k Exp1 >1kb. I have no idea where it come from. I opened this tube only twice, but every time I opened in the hood. I observed a faint band in the reaction containing BSPP Blueprint oligo with slightly larger size.
- I continued to do expansion PCR by using oligo in the reaction 100nM, 2 tubes of 100ul reaction
Expansion PCR
Components | Volume (ul) | Final conc. |
500nM seed oligoes | 40.00 | 100nM |
AP1V6 (100uM) | 0.80 | 400nM |
AP2V6 (100uM) | 0.80 | 400nM |
2x KAPA SYBG fast MM | 100.00 | 1x |
H2O | 58.40 | |
Total | 200.00 |
- Split into 2 tubes (100ul each)
Program 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold ** I stopped at 12 cycles
File:20130530 BSPPBlueprint ExpensionPCR.png
- Purified with Qiaquick column (2 columns) and elute with EB buffer volume 40ul each --> total volume = 80ul
- Measure DNA conc. with Nanodrop: 17.4ng/ul or 175.7nM
- M.W. of dsDNA 150bp = 150bp * 660Da/bp = 99,000 g/mole
- Dilute 1st round amplicon to 10nM volume 400 ul (mix 22.77ul of 175.7nM 1st round amplicons with 377.23 ul H2O) for using as the template for future amplification
- Performed production PCR
Production PCR
- Since I will do trial experiment before do capture on the real samples, I would prepare more probes to make sure that it is enough for entire project.
Components | 1 rxn | 50x rxn mix |
1st round amplicon (10nM) | 0.20 | 10.00 |
AP1V61U (100uM) | 0.40 | 20.00 |
AP2V6 (100uM) | 0.40 | 20.00 |
2x KAPA SYBG fast MM | 50.00 | 2,500.00 |
H2O | 49.00 | 2,450.00 |
Total | 100.00 | 5,000.00 |
- Aliquot 100ul to each tube in 6X 8-strip tubes
Program 95C 30sec -> (95C 10sec -> 55C 20sec-> 72C 30sec) X 15-> 72C 3min -> 15C hold
File:20130530 ProductionPCR BSPPBlueprint.png
- EtOH precipitation of the 48 rxn PCR in 6x 15mL tube
- 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH
- Incubate at -80C O/N -->