Summary
- Restriction Digest/Gel Verification, Concentration Reading
Digest and Gel Verification
| Reagent
|
Volume
|
Expected:
V0120 [no cut sites]
3200 bp
pUC57 [KpnI]
Linearize: 2900 bp
|
20 μL/lane; 1% agarose;
Observed lengths:
W1: Ladder
W2: KAH 021 w/ enzyme
W3: KAH 021 w/o enzyme
W4: KAH 022 w/ enzyme
W5: KAH 022 w/o enzyme
W6: KAH 023 w/ enzyme
W7: KAH 023 w/o enzyme
W8: KAH 024 w/ enzyme
W9: KAH 024 w/o enzyme
Ladder
|
| DNA |
5.0 μL 5.0 μL 8.0 μL 8.0 μL 5.0 μL 5.0 μL 5.0 μL 5.0 μL (From ladder 2 to ladder 9)
|
| 10X buffer |
2.0 μL (for all)
|
| KpnI |
1.0 μL (for even numbered wells only)
|
| dH2O |
8.5 μL 8.5 μL 9.5 μL 9.5 μL 10.5 μL 10.5 μL
|
| Total |
20 μL --> 37°C/ 15 min.
|
Note: This experiment is to verify what backbone the linker is in. There are two wells for each linker, one contains the restriction enzyme KpnI while the other does not. V0120 does not have a cut site for KpnI. If the two wells look the same, the linker is in V0120. If the two wells look different, the linker is in pUC57.
New PcTF Assembly
PCD, AD, RFP in V0120.
Either PCR or cut and paste biobrick assembly.
Biobrick assembly:
PCD: Cut out with Eco&Not
RFP: Cut out with Not&Spe
AD: Cut out with XbaI&PstI
Ligate and then cut with XbaI and SpeI to make compatible with MV10 insertion site.
| 
