High Stabilitiy Plasmid Development
Abstract
- Double Digestions of the plasmids isolated by Fibo yesterday (these).
- Digestions corroboration gel
- False positive detected in the ligation of repC and pSB1AK3
Double Digestions
repC putative ligation
Reactive
|
Digestion
|
Double Dig.
|
Putative ligation plasmid
|
5μL
|
10μL
|
BSA 10x
|
1μL
|
2μL
|
Buffer 2
|
1μL
|
2μL
|
EcoRI-HF
|
1μL
|
1μL
|
SpeI
|
0
|
1μL
|
H2O mQ
|
2μL
|
6μL
|
Total
|
10μL
|
20μL
|
The double digestion must run farther than the simple digestion, and show another band.
B0015 in pSB1AK3
Reactive
|
Stock 2
|
Stock 3
|
B0015 in pSB1AK3
|
22μL
|
22μL
|
BSA 10x
|
3μL
|
3μL
|
Buffer 4
|
3μL
|
3μL
|
EcoRI-HF
|
1μL
|
1μL
|
XbaI
|
1μL
|
1μL
|
H2O mQ
|
0
|
0
|
Total
|
30μL
|
30μL
|
Two more plasmid stocks (1 and 4) were extracted yet not digested.
P1004 in pSB1A1
Reactive
|
Volume
|
P1004 in pSB1A1
|
22μL
|
BSA 10x
|
3μL
|
Buffer 2
|
3μL
|
EcoRI-HF
|
1μL
|
SpeI
|
1μL
|
H2O mQ
|
0
|
Total
|
30μL
|
Gel
Gel ran 40min at 120V, laded with 2μL of dye and 2μL of sample per lane:
Lane
|
Sample
|
1.
|
Ladder 500bp
|
2.
|
Putative ligation of repC in pSB1AK3
|
3.
|
EcoRI-HF linearized put. repC in pSB1AK3
|
4.
|
DD E,S put. repC in pSB1AK3
|
5.
|
B0015 in pSB1AK3 stock 2
|
6.
|
DD B0015 E,X stock 2
|
7.
|
B0015 in pSB1AK3 stock 3
|
8.
|
DD B0015 E,X stock 3
|
9.
|
P1004 in pSB1A1 stock 1
|
10.
|
DD P1004 E,S
|
11.
|
S1 (Dan)
|
12.
|
S2 (Dan)
|
PCR Double Digestions
PCR repC
|
15μL
|
PCR KnR
|
14μL
|
BSA 10x
|
2μL
|
BSA 10x
|
2μL
|
Buffer 2
|
2μL
|
Buffer 2
|
2μL
|
EcoRI-HF
|
0.5μL
|
EcoRI-HF
|
0.5μL
|
SpeI
|
0.5μL
|
SpeI
|
0.5μL
|
H2O mQ
|
0μL
|
H2O mQ
|
1μL
|
Total
|
20μL
|
Total
|
20μL
|
|