PCR of plasmids from Osnat
PCR products
| Plasmid
|
Product
|
Product description
|
Primers
|
Length
|
| ColE2
|
pSG.013
|
Colicin E2 Toxin
|
iSG.003 + iSG.004
|
1797 bp
|
| ColE2
|
pSG.014
|
Colicin E2 Immunity
|
iSG.005 + iSG.006
|
314 bp
|
| ColE7
|
pSG.019
|
Colicin E7 Toxin
|
iSG.001 + iSG.002
|
1784 bp
|
| ColE7
|
pSG.020
|
Colicin E7 Immunity
|
iSG.007 + iSG.008
|
317 bp
|
Primer mix
| 100 uM forward primer
|
10 uL
|
| 100 uM reverse primer
|
10 uL
|
| H2O
|
80 uL
|
Protocol
Prepared in ice:
| H2O
|
37 uL
|
| 5x Phusion HF Buffer
|
10 uL
|
| 10 mM dNTPs
|
1 uL
|
| Primer mix
|
1 uL
|
| Template DNA
|
0.5 uL
|
| Phusion DNA polumerase
|
0.5 uL
|
PCR program
| Cycle step
|
Temperature (C)
|
Time
|
Cycles
|
| Initial denaturation
|
98
|
30s
|
1
|
| Denaturation
|
98
|
5-10s
|
30*
|
| Annealing
|
56
|
10-30s
|
30*
|
| Extension
|
72
|
1m
|
30*
|
| Final extension
|
72
|
5-10m
|
1
|
|
|
4
|
hold
|
1
|
Gel
Ligation
Products
| Product
|
Vector (digested with)
|
Insert (digested with)
|
Description
|
Antibiotic resistance
|
| PSG.001 (-) control
|
PSG.001 (X,P)
|
-
|
PSB3C5 (low to medium copy plasmid)
|
Cm
|
| PSG.002 (-) control
|
PSG.002 (X,P)
|
-
|
PSB4K5 (low copy plasmid)
|
Kan
|
| PSG.005 (-) control
|
PSG.005 (S,P)
|
-
|
pSB3C5 pLac
|
Cm
|
| PSG.006
|
PSG.002 (X,P)
|
PSG.004 (X,P)*
|
pSB4k5 pTet
|
Kan
|
| PSG.008
|
PSG.005 (S,P)
|
PSG.014 (X,P)
|
pSB3C5 pLac ColE2 Imm
|
Cm
|
| PSG.017
|
PSG.001 (X,P)
|
PSG.015 (X,P)*
|
pSB3C5 pConstitutive (0.86)
|
Cm
|
| PSG.018
|
PSG.002 (X,P)
|
PSG.016 (X,P)*
|
pSB4K5 pConstitutive (0.51)
|
Kan
|
*Oligos annealing product
Protocol
|
|
PSG.001 (-) control
|
PSG.002 (-) control
|
PSG.005 (-) control
|
PSG.006
|
PSG.008
|
PSG.017
|
PSG.018
|
| Vector (10-15 ng/uL)
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
| Insert
|
-
|
-
|
-
|
0.2 uL
|
2 uL
|
0.2 uL
|
0.2 uL
|
| 10x T4 DNA Ligase buffer*
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
2 uL
|
| T4 DNA ligase*
|
1 uL
|
1 uL
|
1 uL
|
1 uL
|
1 uL
|
1 uL
|
1 uL
|
| H2O
|
15 uL
|
15 uL
|
15 uL
|
14.8 uL
|
13 uL
|
14.8 uL
|
14.8 uL
|
| Total
|
20 uL
|
20 uL
|
20 uL
|
20 uL
|
20 uL
|
20 uL
|
20 uL
|
*I mixed them in 8x before taking 3uL for each reaction
Let them in 22°C for 1h
Transformation
- Using transformation protocol.
- Using pSG.005 as a positive control.
- Recovery time +/- 1.5h.
- I canceled the pSG.005 and pSG.002 negative control because I mixed them in the same plate.
|
What we did today
