Matt:LabNotes/2016-5-25: Difference between revisions
Jump to navigation
Jump to search
>Mzcai m (→Expansion PCR) |
>Mzcai m (→Expansion PCR) |
||
Line 106: | Line 106: | ||
'''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 | ||
*Purified with Qiagen PCR columns | *Purified with 2 Qiagen PCR columns (75ul PCR product each) | ||
**Elute with 50ul H2O | **Elute with 50ul H2O | ||
*Quantify with Qubit dsDNA | *Quantify with Qubit dsDNA: | ||
**10.1ng/ul / (195bp*607.4Da/bp+157.9Da) = 85.16 nmol/L | |||
*Dilute to 10nM | *Dilute to 10nM | ||
* | **85.16nM x 99ul = 10nM x 843ul | ||
**Add 744ul H2O | |||
===Production PCR=== | |||
*V4 master mix | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (50X)''' | |||
|- | |||
| First round amplicon TB12k_Apr2016_V4 (10nM)||0.2||10 | |||
|- | |||
| 2X KAPA SYBG MM||50||2500 | |||
|- | |||
| AP1V4U (100uM)||0.4||20 | |||
|- | |||
| AP2V4 (100uM)||0.4||20 | |||
|- | |||
| H2O||49||2450 | |||
|- | |||
| Total||100||5000 | |||
|} | |||
'''Program''' 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold | |||
*Stopped after cycles | |||
[[File:.JPG | 800px]] |
Revision as of 19:47, 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
- Purified with 2 Qiagen PCR columns (75ul PCR product each)
- Elute with 50ul H2O
- Quantify with Qubit dsDNA:
- 10.1ng/ul / (195bp*607.4Da/bp+157.9Da) = 85.16 nmol/L
- Dilute to 10nM
- 85.16nM x 99ul = 10nM x 843ul
- Add 744ul H2O
Production PCR
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon TB12k_Apr2016_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
- Stopped after cycles