Plasmid minipreb for ClpX, ClpB and ChiT

4/17/2015

Clones:
ClpX 1,2,3,4
ClpB 1,2,3,4
ChiT 1,2,3,4
Clones fromTransformation using chemically competent cells for ClpX,ClpB and ChiT + pJH1

Zyppy™ Plasmid Miniprep Kit was used Protocol

SOE PCR for clpX, ClpB and ChiT

4/10/2015

1-
1.5  μL ClpX 3`           sample 1,     2/2/2015
1.5  μL ClpX 5`           sample 2,     2/11.2015  
1.5  μL ClpX 3` R primares
1.5  ClpX 5` F primares
20 micro supermix.
2-
1.5  μL ClpB 3`           sample 1,     2/8/2015
1.5  μL ClpB 5`           sample 3,     2/11.2015  
1.5  μL ClpB 3` R primares
1.5  ClpB 5` F primares
20 micro supermix.

3-
1.5  μL ChiT 3`           sample ,     3/3/2015
1.5  μL ChiT 5`           sample 4,     2/11.2015  
1.5  μL ChiT 3` R primares
1.5  ChiT 5` F primares
20 micro supermix.

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.

Transformation using chemically competent cells for ClpX,ClpB and ChiT + pJH1

4/1/2015
ClpX,ClpB and ChiT + pJH1

One Shot® Mach1™-T1R Chemically Competent E. coli
Transforming Competent Cells Perform the following before starting the transformation procedure:
 • Equilibrate a water bath to 42°C. 
• Warm the vial of S.O.C. Medium (supplied with the kit) to room temperature. 
• Spread X-Gal onto LB agar plates containing antibiotic, if desired. 
• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). 
If you are including the pUC19 control, make sure that you have one LB agar plate containing 100 µg/ml ampicillin.

 Note: For optimal growth of Mach1™-T1R E. coli cells, it is essential that selective plates are prewarmed to 37°C prior to spreading. Transformation Procedure We recommend including the pUC19 control plasmid DNA supplied with the kit in your transformation experiment to verify the efficiency of the competent cells.

1. Thaw, on ice, one vial of One Shot® Mach1™-T1R Chemically Competent E. coli for each transformation. 
2. Add 1 to 5 µl of the DNA (10 pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. If you are transforming the pUC19 control, add 1 µl (10 pg) into a separate vial of One Shot® cells and mix gently.
 3. Incubate the vial(s) on ice for 30 minutes.
 4. Heat-shock the cells for 30 seconds at 42°C without shaking. 
5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
 6. Add 250 µl of room temperature S.O.C. Medium to each vial. 
7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.
 8. Spread 25-100 µl of the transformation mix on a prewarmed selective plate. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired. 
9. Invert the plate(s) and incubate at 37°C. If you are using ampicillin selection, visible colonies should appear within 8 hours, and blue/white screening can be performed after 12 hours. If you are selecting transformants with an antibiotic other than ampicillin, incubate plates overnight.
 10. Select overnight-grown colonies and analyze by plasmid isolation, PCR, or sequencing.
 For plasmid isolation, inoculate a single, overnight-grown colony in 2 ml of prewarmed selective media .
 For optimal results, we recommend inoculating as much of the single colony as possible. Shake at 37°C for 4 hours before isolating the plasmid. 

Result
1-ClpX  + pJH1 ,on LB+ Hygromycine (+)
2-ClpB  + pJH1 , on LB+ Hygromycine(+)
3- ChiT + pJH1, on LB+ Hygromycine (+)
4- PUC 19 on LB+ Ampenicillin, (+)  positive control.
5- DH5 alfa on LB+ Ampenicillin (-) negative control.
6-PUC 19 and DH5 alfa on LB+ Hygromycine (-)

Ligation SOE PCR with pJH1

3/30/2015
ClpX, ClpB (insert)
pJH1 ,Δ dephosphatase and Δ SmaI (vector)
ClpX Insert 3 μL : pJH1 1 μL + 6 dH2O = total 10  μL 
ClpB Insert 3 μL : pJH1 1 μL+ 6 dH2O = total 10  μL  , brief centerfugation 

Ligation using .neb.com/protocols

Quick Ligation Protocol (M2200)

Buffers

Quick Ligation Reaction Buffer

Protocol

1. Combine 50 ng of vector with a 3-fold molar excess of insert. Use NEBioCalculator to calculate molar ratios. Adjust volume to 10 μl with dH₂O.
2. Add 10 μl of 2X Quick Ligation Buffer and mix.
3. Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
4. Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
5. Chill on ice, then transform or store at -20°C.
6. Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.

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