End repair for SOE (Clpx,ClpB and Chit) PCR.

3/6/2015
ClpX

• DNA                        12 μL 

• End repair enzyme (0.5μL  )  from life tech.

• Buffer                      ( 2 μL )

• dH2O                      ( 5 μL )

ClpB

• DNA                        20 μL 

• End repair enzyme (0.5μL  )

• Buffer                      ( 2 μL )

Chit

• DNA                        20 μL 

• End repair enzyme (0.5μL  )

• Buffer                      ( 2 μL)

Incubate 37 °C    for 15 minutes, inactivation 75  °C   for 10 minutes.

Enzyme digest and treatment pJH1 with SmaI, and Antarctic Phosphatase

3/13/2015

1-In a 1.5mL tube combine the following:

• DNA     15 μL (1-5 mg) 

• Restriction Enzyme(1μL SmaI )

• Buffer_cutsmart_( 2 μL )

• dH2O ( 2 μL )

              20 μL total.Incubate 25 °C    for 2 hours, inactivation 65  °C   for 20 minutes.
2-Antarctic Phosphatase catalyzes the removal of 5´ phosphate from DNA.

• DNA     15 μL (from #1 above Δ SmaI) 

• Antarctic Phosphatase(1μL SmaI )

• Buffer ( 2 μL )

• dH2O ( 2 μL )

              20 μL total.Incubate 37 °C    for 15 minutes, inactivation 70  °C   for 5 minutes.

Plasmid miniprep. pJH1

3/2/2015
E.coli with pJH1 grew on LB+ Hygromycine 250 mg/ml, after 24h incubation at 37 C a single colony was inculated into 150 ml LB broth + Hygromycine 250 mg/ml.

FosmidMAX™ DNA Purification Kit Cat. No. FMAX046 used Protocol

1% agarose

pJH1

                                                     

PCR for LVS chiT3 .

3/3/2015

Protocol 

95 c                    3 minutes

95 c                    30 second

41 c                     1.5 minutes                                35 cycle

68 c                    1.5 minute
Final elongation 5 minutes, total volume 25 microliter.

Samples:
1-chiT3

1.5 mico primares x 2
1.5
micro g DNA
20 micro supermix.
Run on 1% gel for 3 hours.

SOE PCR for clpB

clpB3 R primer 1.5 μL
clpB5 F primer 1.5 μL
supermix 21 μL


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.

1% agarose run for 4 hours.

SOE PCR for clpX

2/23/2015

3 PCR 1.5 μL (PCR product from clpX 3)
5 PCR 1.5  μL (PCR product from clpX 5)
clpX3 R primer 1.5 μL
clpX5 F primer 1.5 μL
supermix 21 μL


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.

1% agarose run for 4 hours.

PCR for LVS chiT3, clpX3,, clpB5 and chiT5

2/11/2015

Protocol 5

95 c                    3 minutes

95 c                    30 second

44 c                     1.5 minutes                                35 cycle

68 c                    1.5 minute
Final elongation 5 minutes, total volume 25 microliter.

Samples:
1-chiT3
2-clpX3
3- clpB5
4
- chiT5
1.5 mico primares x 2
2 micro g DNA
20 micro supermix.

Run on 1% gel for 3 hours.