01/22/15
Planning: Gal4 fusion cloning
- "Backbone" = Gal4-mCherry-VP64 construct KAH60/pcVN
- Approach: swap-in activator domain to replace VP64
- PCR-amplify backbone (omitting VP64)
- PCR-amplify insert
- Plan A: use LCR to ligate insert into backbone
Primers (ordered 01/02/15)
Attempt 1: swap-in ATF activation domain; add 5' phosphates to all oligos EXCEPT for bridging oligos
- KAH60 "backbone" (without VP64, includes scars) = 6637
- ATF2 insert = 906
- LCR Bridging oligos
- LCRb_KAH60_ATF2_1
- LCRb_KAH60_ATF2_2
PCR-verify fragment primers
Try different cDNA libraries for the ATF2 fragment to account for any mutations or deletions
- KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1
- U2OS C002; ATF2 insert = 906: ATF2_f1/ ATF2_r1
- SKNSH C001; same
- K562 C001; same
Reagent
|
Rxn1
|
Rxn2
|
Rxn3
|
Rxn4
|
30 μL/lane, 1% agarose; Ladder
|
Template |
0.2 KAH60/pcVN |
1.0 1:1000 U2OS cDNA |
1.0 1:1000 SKNSH cDNA |
1.0 1:1000 K562 cDNA
|
10 uM fwd primer |
1.0 |
1.0 |
1.0 |
1.0
|
10 uM rev primer |
1.0 |
1.0 |
1.0 |
1.0
|
2x GoTaq green |
12.5 |
12.5 |
12.5 |
12.5
|
dH2O |
10.3 |
9.5 |
9.5 |
9.5
|
Program: GOTAQ35cyc
- 95°C, 3 min
- 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
- 72°C, 3 min
- 4°C ∞
|