Matt:LabNotes/2016-5-25: Difference between revisions
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95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold | 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold | ||
[[ | | [[File:20160526_TB12k_ExpansionPCRtest.JPG | 450px]] | ||
*1,000X final dilution was in the sweet spot for number of cycles (15-20) | *1,000X final dilution was in the sweet spot for number of cycles (15-20) | ||
*Use that concentration and do 19 cycles | *Use that concentration and do 19 cycles | ||
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| Total||150 | | Total||150 | ||
|} | |} | ||
*50ul per PCR tube | |||
'''Program''' | '''Program''' | ||
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold | 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold |
Revision as of 00:26, 27 May 2016
TB12k_Apr2016 Probe Preparation
- Probe Design
- Received 195-mer oligos 5/25/26
- Name: TB12k_Apr2016_DARTFISH
- Amount: 341ng
- Physical State: Solid
- Calculate MW of 195 nt probe = 195nt * 303.7Da/nt + 79Da = 59,300.5 g/mol
- 341ng -> 5.75pmol
Resuspend and Aliquot
- Resuspend in 100ul (57.5nM)
- Pipetted 20X to mix
- Made aliquots of 40, 40, 10, and 10 into 0.5ml Lo-Bind tubes
Serial Dilution 1000X
- Take 1ul from a 10ul aliquot and add 9ul H2O (10X Dilution)
- Take 5ul and add 45ul H2O (100X Dilution)
- Take 20ul and add 180ul H2O (1000X Dilution)
Expansion PCR Test
- NTC
Component | Volume |
Seed oligo | 0 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 10.5 |
Total | 25 |
- Positive Control
Component | Volume |
CA12kNov2014_V4 1st rnd Oligos (10nM) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
- 1,000X Dilution
Component | Volume |
Seed oligo (100X Dilution) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
- 10,000X Dilution
Component | Volume |
Seed oligo (1000X Dilution) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold
File:20160526 TB12k ExpansionPCRtest.JPG
- 1,000X final dilution was in the sweet spot for number of cycles (15-20)
- Use that concentration and do 19 cycles
Expansion PCR
Component | Volume |
Seed oligo (100X Dilution) | 15 |
F/R V4 Primer Mix (10uM) | 12 |
2X Kapa MM | 75 |
H2O | 48 |
Total | 150 |
- 50ul per PCR tube
Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold