Noi/NOTES/2013-5-30: Difference between revisions
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** Volume 80ul in TE buffer. Total amount 3.21ug. | ** Volume 80ul in TE buffer. Total amount 3.21ug. | ||
* Probe prep plan | * Probe prep plan | ||
** Last time of Matt's experiment on Expansion PCR of oligo from CustomArray, we need to used template ~ | ** 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 | ** Aliquot 40ul oligo from original tube and dilute to 500nM (add 24.90ul H2O to 40ul of 811.3nM oligo) | ||
== Expansion PCR (TEST) == | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''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| 200px]] | |||
* 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 | |||
# 10bp DNA ladder | |||
# O: 3ul of 500nM oligo before expansion PCR | |||
# PC (BSPP DMR330k Exp1) | |||
# 20nM BSPP _Blueprint after expaion PCR | |||
# 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 == | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''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 |
Revision as of 23:57, 30 May 2013
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
- 10bp DNA ladder
- O: 3ul of 500nM oligo before expansion PCR
- PC (BSPP DMR330k Exp1)
- 20nM BSPP _Blueprint after expaion PCR
- 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