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= Expansion PCR of new probe set from CustomArrays (90k_oligos_30Sept2014) = | |||
* [[noi:DMR220k_LabNotes| '''Link to calendar''']] | * [[noi:DMR220k_LabNotes| '''Link to calendar''']] | ||
* 2014-10-15: Received :90k_oligos_30Sept2014 from CustomArray | * 2014-10-15: Received :90k_oligos_30Sept2014 from CustomArray | ||
Line 4: | Line 5: | ||
* Name: 90k_oligos_30Sept2014 | * Name: 90k_oligos_30Sept2014 | ||
* Conc. 69.89ng/ul, | * Conc. 69.89ng/ul, | ||
* Volume 80ul in TE buffer. Total amount | * Volume 80ul in TE buffer. Total amount 5.59ug | ||
* [[Kun:LabNotes/MONOD/2014-8-4#Re-design_of_selector_probes| Info from Dr. Zhang's note]] | * [[Kun:LabNotes/MONOD/2014-8-4#Re-design_of_selector_probes| Info from Dr. Zhang's note]] | ||
*Sept14 probe set: [[Media:90k_oligos_30Sept2014.txt.gz]] | *Sept14 probe set: [[Media:90k_oligos_30Sept2014.txt.gz]] | ||
Line 24: | Line 25: | ||
H2O 4.01 | H2O 4.01 | ||
Total 10.00 | Total 10.00 | ||
* 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X | * 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 17-> 72C 3min -> 15C hold | ||
[[File:2014-10-15_testExpPCR_Sep14probes.PNG| 500px]] | |||
* The qPCR curves of all reactions showed amplification very well ~16 cycles for the new probe set. I then continued to do expansion PCR in larger volume (150ul, 100uM of the template for each subset) without gel verification | * The qPCR curves of all reactions showed amplification very well ~16 cycles for the new probe set. I then continued to do expansion PCR in larger volume (150ul, 100uM of the template for each subset) without gel verification | ||
* <span style="color:red">I SHOULD HAVE VERIFIED AMPLICON SIZE IN TBE GEL BEFORE CONTINUE TO DO EXPANSION PCR IN A LARGE VOLUME EVEN THE qPCR CURVE SHOWED AMPLIFICATION AS USUAL.</span> | |||
'''Components Volume (ul)''' | '''Components Volume (ul)''' | ||
Seed oligo (1694.3nM) 8.85 | Seed oligo (1694.3nM) 8.85 | ||
F primer | F primer (10uM) 0.60 | ||
R primer | R primer (10uM) 0.60 | ||
2x KAPA SYBG fast MM 75.00 | 2x KAPA SYBG fast MM 75.00 | ||
H2O 64.95 | H2O 64.95 | ||
Line 35: | Line 39: | ||
* Split each set in 3X50ul | * Split each set in 3X50ul | ||
* 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X '''16'''-> 72C 3min -> 15C hold | * 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X '''16'''-> 72C 3min -> 15C hold | ||
[[File:2014-10-15_ExpPCR_Sep14probes_LMScluster-padlockSNP.png|500px]] | |||
* Purify the 1st amplicons with 2x QIAquick column and elute with 50ul TE buffer | * Purify the 1st amplicons with 2x QIAquick column and elute with 50ul TE buffer | ||
* Measure concentration by N.D. | * Measure concentration by N.D. | ||
* Will add N.D. result. | * Will add N.D. result. | ||
* I then continue to amplify cancer_hyb_sep14 probe set with the same condition of the quick test for the two subsets above for | * I then continue to amplify cancer_hyb_sep14 probe set with the same condition of the quick test for the two subsets above for 15 cycles. The amplification worked fine at 15 cycles. | ||
[[File:2014-10-15_testExpPCR_Sep14probes_v8.png|500px]] | |||
* I ran all 1st round amplicon in 6% TBE gel | * I ran all 1st round amplicon in 6% TBE gel | ||
** For quick test PCR product, I added 2ul of 6X loading dye to 10ul of PCR product and loaded 3ul in the gel. For column purified amplicons, I loaded 1ul for each | ** For quick test PCR product, I added 2ul of 6X loading dye to 10ul of PCR product and loaded 3ul in the gel. For column purified amplicons, I loaded 1ul for each | ||
[[File:ZhangLab_2 2014-10-16 14hr 04min_test_ExpPCR_Verify.jpg| 400px]] | [[File:ZhangLab_2 2014-10-16 14hr 04min_test_ExpPCR_Verify.jpg| 400px]] | ||
LMS = LMS_selector | |||
padl = padlock_SNP | |||
PTC = positive control | |||
canc = cancer_hyb_sep14 | |||
* From the gel image, there is a smear below 100bp band in all the three subsets, which is unexpected. The amplicons size should be ~125-130bp. The sizeof positive control are correct. I am pretty sure that I added the right reagents in each reaction. In addition, the result of expansion PCR in large volume of LMS_selector and padlock_SNP were consistent with the quick test PCR. | |||
* I will check the result with Chris for his probe set to see if he has the same issue. | |||
** [[Chris:LabNotes/FateMapping/Calendar/2014/2014-10-16| '''Chris's result''']]<br> | |||
* From Chis's result, he got the similar smear below 100bp like the other three subsets amplified by me. | |||
* I double-check the primer/adaptor sequences of the designed probes. The design is correct for the four subsets and the number of each subset agrees with Dr. Zhang's note. | |||
** [[Media:2014-10-16-90k-sep14-primer-check.pdf|'''Primer/adaptor sequence check''']] | |||
* Dr. Zhang notes that CustomArray has started to synthesize the new batch of these probe sets. |
Latest revision as of 12:34, 24 December 2014
Expansion PCR of new probe set from CustomArrays (90k_oligos_30Sept2014)[edit]
- Link to calendar
- 2014-10-15: Received :90k_oligos_30Sept2014 from CustomArray
Oligo info.[edit]
- Name: 90k_oligos_30Sept2014
- Conc. 69.89ng/ul,
- Volume 80ul in TE buffer. Total amount 5.59ug
- Info from Dr. Zhang's note
*Sept14 probe set: Media:90k_oligos_30Sept2014.txt.gz probe set # probes Length Amp primers Chris gDNA_MS_v2 8,045 127bp V6(G*T*CATATCGGTCACTGTU//5Phos/GGGTAGTGTGTATCCTG) Kun cancer_hyb_sept14 51,639 110-130bp V8(T*C*TAATCTAGCGCGACGTCU//5Phos/CCACAAGAGGCGCTATG) Kun LMS_selector 17,342 124-130bp NE(TGCCTAGGACCGGATCAACT/GCTTCGGTTCACGCAATG) Kun padlock_SNPs 12,974 125bp V4(G*A*CTGGAAGAGCACTGTU//5Phos/AGCCTCATGCGTATCCG) Total 90,000
- Length: I would use the average size of the oligo pool to calculate concentration ~125nt = MW=41250g/mole
- Conc. : 69.89 ng/ul = 1694.30 (69.89ng/ul/ 41250g/mole)
Expansion PCR (Quick test, round1)[edit]
- I initially work on the two subsets, including LMS_selector (NE or eMIP_CA1 primer set) and padlock_SNP (V4 primer set)
- I will include the two positive control for the two primer set to confirm that the amplification works fine. Note that I combine F & R primer in final conc. 10uM in the same tube.
Components Volume (ul) Final conc. Seed oligo (1694.3nM) 0.59 100nM F/R primer mix (10uM) 0.40 400nM 2x KAPA SYBG fast MM 5.00 1x H2O 4.01 Total 10.00
- 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 17-> 72C 3min -> 15C hold
File:2014-10-15 testExpPCR Sep14probes.PNG
- The qPCR curves of all reactions showed amplification very well ~16 cycles for the new probe set. I then continued to do expansion PCR in larger volume (150ul, 100uM of the template for each subset) without gel verification
- I SHOULD HAVE VERIFIED AMPLICON SIZE IN TBE GEL BEFORE CONTINUE TO DO EXPANSION PCR IN A LARGE VOLUME EVEN THE qPCR CURVE SHOWED AMPLIFICATION AS USUAL.
Components Volume (ul) Seed oligo (1694.3nM) 8.85 F primer (10uM) 0.60 R primer (10uM) 0.60 2x KAPA SYBG fast MM 75.00 H2O 64.95 Total 150.00
- Split each set in 3X50ul
- 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 16-> 72C 3min -> 15C hold
File:2014-10-15 ExpPCR Sep14probes LMScluster-padlockSNP.png
- Purify the 1st amplicons with 2x QIAquick column and elute with 50ul TE buffer
- Measure concentration by N.D.
- Will add N.D. result.
- I then continue to amplify cancer_hyb_sep14 probe set with the same condition of the quick test for the two subsets above for 15 cycles. The amplification worked fine at 15 cycles.
File:2014-10-15 testExpPCR Sep14probes v8.png
- I ran all 1st round amplicon in 6% TBE gel
- For quick test PCR product, I added 2ul of 6X loading dye to 10ul of PCR product and loaded 3ul in the gel. For column purified amplicons, I loaded 1ul for each
File:ZhangLab 2 2014-10-16 14hr 04min test ExpPCR Verify.jpg LMS = LMS_selector padl = padlock_SNP PTC = positive control canc = cancer_hyb_sep14
- From the gel image, there is a smear below 100bp band in all the three subsets, which is unexpected. The amplicons size should be ~125-130bp. The sizeof positive control are correct. I am pretty sure that I added the right reagents in each reaction. In addition, the result of expansion PCR in large volume of LMS_selector and padlock_SNP were consistent with the quick test PCR.
- I will check the result with Chris for his probe set to see if he has the same issue.
- From Chis's result, he got the similar smear below 100bp like the other three subsets amplified by me.
- I double-check the primer/adaptor sequences of the designed probes. The design is correct for the four subsets and the number of each subset agrees with Dr. Zhang's note.
- Dr. Zhang notes that CustomArray has started to synthesize the new batch of these probe sets.