04/14/15
LCR Protocol Development
- Refer to entry 04/06/2015
- Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)
- Gal4DB-mCh (1170 bp): Template - KAH228 plasmid, F - Gal4DB f1 (Tm = 47), R - mCh r1 (Tm = 56)
- ATF2 (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1 (Tm = 56) , R - ATF2_r1 (Tm = 58)
- MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1 (Tm = 64), R - MV10 r1 (Tm = 69)
MV10 - Gradient PCR to enhance 5 kb band
- 68°C
- 67.3°C
- 65.9°C
- 63.9°C
- 61.4°C
- 59.6°C
- 58.1°C
- 57°C
Reagent
|
Rxn1-8
|
(x9)
|
Expected: 1-8. MV10 = 5191
|
10 μL/lane, 1% agarose; Ladder
|
Template |
0.2 |
1.8
|
10 uM fwd primer |
1.0 |
9.0
|
10 uM rev primer |
1.0 |
9.0
|
10 mM dNTPs |
1.0 |
9.0
|
Phusion pol. |
0.5 |
4.5
|
5x HF buffer |
5.0 |
45.0
|
dH2O |
41.3 |
371.7
|
|
50.0
|
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
- 98°C, 3 min
- 35x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Failed
- Try again with 5x GC buffer (more robust amplification) and DMSO (template is 53.7 GC)
Gal4DB-mCh - plasmid
ATF2 - 1:100 iPSC cDNA
Reagent
|
Rxn1,2
|
Rxn3,4
|
Expected: 1,2. Gal4DB-mCh = 1170 3,4. ATF2 = 906
|
10 μL/lane, 1% agarose; Ladder
|
Template |
0.2 |
3.0
|
10 uM fwd primer |
1.0 |
1.0
|
10 uM rev primer |
1.0 |
1.0
|
10 mM dNTPs |
1.0 |
1.0
|
Phusion pol. |
0.5 |
0.5
|
5x HF buffer |
5.0 |
5.0
|
dH2O |
41.3 |
38.5
|
|
50.0 |
50.0
|
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 30 sec]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Gal4DB-mCh - success!
- purify with Sigma PCR purification kit
- Elute & back-elute w/ 25 μL elution sln.
- ATF2 - failed
- Try again with 1:10 and undiluted template
- Use 5x GC buffer to boost amplification
Measure conc.'s - Gal4DB-mCh PCR
Sample |
OD260 |
260/280 |
ng/μL
|
1. 5'p-Gal4DB-mCh |
0.01 |
1.23 |
9.68
|
MV10 Trial 2 - Gradient PCR to enhance 5 kb band
- 68°C
- 67.3°C
- 65.9°C
- 63.9°C
- 61.4°C
- 59.6°C
- 58.1°C
- 57°C
Reagent
|
Rxn1-8
|
(x9)
|
Expected: 1-8. MV10 = 5191
|
10 μL/lane, 1% agarose; Ladder
|
Template |
0.2 |
1.8
|
10 uM fwd primer |
1.0 |
9.0
|
10 uM rev primer |
1.0 |
9.0
|
10 mM dNTPs |
1.0 |
9.0
|
Phusion pol. |
0.5 |
4.5
|
5x GC buffer |
5.0 |
45.0
|
DMSO |
1.5 |
7.5
|
dH2O |
39.8 |
358.2
|
|
50.0
|
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
- 98°C, 3 min
- 35x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Failed - try using XbaI-linearized vector as template
- XbaI cuts in between the two primers
ATF2
- 1:1 iPSC cDNA
- "
- 1:10 iPSC cDNA
- "
Reagent
|
Rxn1,2
|
Rxn3,4
|
Expected: 1,2. Gal4DB-mCh = 1170 3,4. ATF2 = 906
|
No visible product in any lanes
|
Template |
0.5 |
2.0
|
10 uM fwd primer |
1.0 |
1.0
|
10 uM rev primer |
1.0 |
1.0
|
10 mM dNTPs |
1.0 |
1.0
|
Phusion pol. |
0.5 |
0.5
|
5x GC buffer |
5.0 |
5.0
|
dH2O |
41.0 |
39.5
|
|
50.0 |
50.0
|
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 30 sec]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Failed - order ATF2 cDNA, use as new template for PCR
|