Revision as of 19:47, 27 May 2016 by >Mzcai
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)
- 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
Component
|
Volume
|
Seed oligo |
0
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
10.5
|
Total |
25
|
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
|
Component
|
Volume
|
Seed oligo (100X Dilution) |
2.5
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
8
|
Total |
25
|
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
|
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)
- 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
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
File:.JPG