Noi/NOTES/2014-8-21: Difference between revisions
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** Subset B: 150nM template, 16 and 17 cycles | ** Subset B: 150nM template, 16 and 17 cycles | ||
** Subset C: 150nM and 200nM. 18 cycles | ** Subset C: 150nM and 200nM. 18 cycles | ||
[[File:2014-08-21_test_ExpPCR-90k_15x.PNG| 500px]] [[File:2014-08-21_test_ExpPCR-90k_16x.PNG| 500px]] | |||
[[File:2014-08-21_test_ExpPCR-90k_17x.PNG| 500px]] [[File:2014-08-21_test_ExpPCR-90k_18x.PNG| 500px]] | |||
* Loaded 5ul of amplicons from each subset at different PCR conditions and also re-loaded amplicons from the 1st test in 6% TBE gel | * Loaded 5ul of amplicons from each subset at different PCR conditions and also re-loaded amplicons from the 1st test in 6% TBE gel | ||
[[File:ZhangLab_2 2014-08-21 18hr 58min_90k-testExpPCR-r2.jpg|500px]] | [[File:ZhangLab_2 2014-08-21 18hr 58min_90k-testExpPCR-r2.jpg|500px]] |
Revision as of 15:43, 22 August 2014
- Link to calendar
- 2014-08-13: Received MOMOD V4 Oligo pool from CustomAraay
Oligo info.
- Name: 90K_ologos_04Aug2014 (8-20-14)
- Conc. 153.49ng/ul,
- Volume 80ul in TE buffer. Total amount 12.28ug
- Info from Dr. Zhang's note
Description # oligos Length Amp. Primers Adaptor Removal Set A: BSPP LMS cluster probes 73,574 148nt V6 USER/DpnII Set B: Selector LMS cluster 12,994 148nt V4 USER/DpnII Set C: Selector chr21 unique 2,539 148nt eMIP_CA_F/R Nt.AlwI/Nb.BrsDI Total: 89,107
- I will be working on 73K probe set
- Length 148 mer (MW = 148 * 330Da/bp = 48,840 Da or g/mole
- Conc. 20.53ng/ul = 3.1427uM or 3142.7nM (153.49ng/ul/ 48,840g/mole)
Expansion PCR (Quick test, round1)
- I did a quick test of expansion PCR on the three subset (20ul reaction).
- I diluted 1st amplicon of previous probe set amplified by each primer set (by V4, V6, or eMIP_CA primers) into 2nM and will be as positive control for amplification.
- V4 set: I used MONOD GP1V4 as template. Mix 10ul of 20nM 1st round amplicon MONOD GP1V4 with 90ul H2O
- V6 set: I used MONOD V3 as template. Prepare 2nM template in the same way as V4 set.
- eMIP_CA: I used oligo from LC sciences used in Nature methods 2012. Mix 20ul of 10nM 1st round amplicon 4K LC Sciences with 80ul H2O
95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 18-> 72C 3min -> 15C hold
File:2014-08-21 test ExpPCR-90k 18x-r1.PNG
- Loaded 3ul of positive control and 5ul of amplicons from each subset in 6% TBE gel
File:ZhangLab 2 2014-08-21 15hr 36min 90K-testExpPCR.jpg
- I switched the labeling order. It should be Set A, Set B and Set C
Subset | # oligos | % in the pool |
Set A | 73,574 | 82.57 |
Set B | 12,994 | 14.58 |
Set C | 2,539 | 2.85 |
- Based on qPCR curve and gel image of the largest subset, subset A (82.57%), it looked like the amplification efficiency was not as high as previous batches of oligos from CustomArray. For optimization of expansion PCR in Blueprint project and other batches of oligos from CustomArray, qPCR curve started to shoot up ~6-7 cycles for 100nM oligo template and the band intensity was stronger by loading only 2.5ul of amplicons.
- The smaller subset (set B and set C) had very faint intensity, so Rui wanted to know if increasing template amount would increase the intensity of the 150bp amplicons without inhibiting PCR reaction. I then tried a quick PCR again by increasing template concentration of set B to 150nM and reduce PCR cycle to 16 and 17 cycles. For subset C, I increase template concentration to 150nM and 200nM and ran PCR for 18 cycles since it takes only 2.85% of the pools. For subset A, I reduced PCR cycle number to 15 and 16 cycles, but used the same concentration of 100nM.
Result of expansion PCR in Blueprint project
File:20130530 BSPPBlueprint TESTExpensionPCR.png File:ZhangLab 2 2013-05-30 15hr 54min TESTExpansionPCR.jpg File:20130530 BSPPBlueprint ExpensionPCR.png
Expansion PCR (Quick test, round2)
- As discussed above, I did a quick run (10ul reaction) with following condition
- Subset A: 100nM template, 15 and 16 cycles
- Subset B: 150nM template, 16 and 17 cycles
- Subset C: 150nM and 200nM. 18 cycles
File:2014-08-21 test ExpPCR-90k 15x.PNG File:2014-08-21 test ExpPCR-90k 16x.PNG File:2014-08-21 test ExpPCR-90k 17x.PNG File:2014-08-21 test ExpPCR-90k 18x.PNG
- Loaded 5ul of amplicons from each subset at different PCR conditions and also re-loaded amplicons from the 1st test in 6% TBE gel