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 ~50x 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 useDMR330k oligos (Exp1) from Agilent as PC.
    • Aliquot 40ul oligo from original tube and dilute to 500nM (add 24.90ul H2O to 40ul of 811.3nM oligo)

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
  • 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

File:ZhangLab 2 2013-05-30 15hr 54min TESTExpansionPCR.jpg

  • 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
  1. 10bp DNA ladder
  2. O: 3ul of 500nM oligo before expansion PCR
  3. PC (BSPP DMR330k Exp1)
  4. 20nM BSPP _Blueprint after expaion PCR
  5. 50nM BSPP _Blueprint after expaion PCR
  • From the gel image above, the expected band 0f ~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 contain 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 tube (100ul each)
Program

95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold

** I stopped at 12 cycles
  • Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
  • Measure DNA conc. with Nanodrop: 13.2 ng/ul or 103nM
  • M.W. of dsDNA 150bp = 150bp * 660Da/bp = 99,000 g/mole

13.2ng/ul size 210bp = 103nM

  • Dilute 1st round amplicon to 10nM volume 400 ul (mix 38.83 ul of 103nM 1st round amplicons with 361.17 ul H2O): for using as the template for the future amplification
  • Perform production PCR