SOE PCR new prtocol 26_5_2015

5/26/2015
1-
1  μL      3` R primares
1  μL      3` F primares
2 μL        PCR(ClpB) from 4/10/2015
12.5 μL   supermix.
8 μL dd water.

Protocol:
 95 °C    3 minutes

95 °C   30 seconds
72 °C  2 minutes                    35 cycles
68 °C  1.5 minutes

Final elongation 68 °C  for 5 minutes.

2-
1  μL      5` R primares
1  μL      5` F primares
2 μL        PCR(ClpB) from 4/10/2015
12.5 μL   supermix.
8 μL dd water.

Protocol:
 95 °C    3 minutes

95 °C   30 seconds
72 °C  2 minutes                    35 cycles
68 °C  1.5 minutes

Final elongation 68 °C  for 5 minutes.

2-ClpB 5 prim(500bp)
empty
1-ClpB3 prim(500bp)

PCR for plasmidminipreb

5/20/2015
Plasmids from Plasmid minipreb for ClpX, ClpB and ChiT

Negative control: pJH1 with ClpB primares.

1% agrose run for 2 h.

ChiT4     (band)
ChiT3     (band)
ChiT2
ChiT1
ClpB4
B3
B2
ladder
empty
B1        (band)
pJH1
ClpX4
X3
X2
X1
ladder

Direct PCR for the insert clones

5/6/2015
Clones were grown on LB+ Hygromycin 250 mg/ml, direct sample was taken from the colony.

• Clones: ClpX1, X2, X3, X4, X5, X6, X7,X8 ,X9 ,X10 and X11  
• ClpX primar with pJH1 clone as a control.
• ClpX PCR as a positive control.

• ClpB1, B2,B3, B4, B5, B6, B7and B8 .
• ClpB primar with pJH1 clone as a control.
• ClpB PCR as a positive control.

• ChiT1,T2,T3 and T4 
•  ChiT primar with pJH1 clone as a control,ChiT primar with B/r E. coli clone as a control. 
• ChiT PCR as a positive control.

1  μL      3` R primares
1  μL      5` F primares
12.5 μL   supermix.
10 μL dd water.

Protocol:
 95 °C    3 minutes

95 °C   30 seconds
55 °C  2 minutes                    30 cycles
68 °C  1.5 minutes

Final elongation 68 °C  for 5 minutes.

ChiT B/r
ChiT,pJH1
ChiT PCR 
ChiT3

ChiT2
ChiT1



ClpB,pJH1
ClpB,PCR
ClpB3
ClpB2
ClpB1
ClpX,pJH1
ClpX,PCR
ClpX3
ClpX2
ClpX1

ladder