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Objective
Prepping some work for the weeks ahead
Cloning
PCR
- I'm going to try to do the Mb cloning into pQE-80 myself. (May want to try pDrive Coning Vector eventually)
- The following protocol is taken from here
- PCR mix
- 10X reaction Buffer
- 2.5μL
- NTP's stock solution is 10mM (make 50μL of 2mM by diluting 10μL of stock)
- 2.5μL
- 3' primer (25μM) (need to check my concentrations for rough estimates)
- 1.0μL
- 5' primer (25μM) (need to check my concentrations for rough estimates)
- 1.0μL
- DNA template (0.1μg/μL) (need to check my concentration for rough estimate)
- 1.0μL
- Autoclaved water
- 16.5μL
- Pfu polymerase
- 0.5μL
- PCR cycles
- 1' 94°C
- 1' 55°C
- 1' 72°C
- Repeat previous three steps 3 times
- 1' 94°C
- 1' 60°C
- 1' 72°C
- Repeat previous three steps 12 times
- 7' 72°C
Purifying PCR product
- Run a 1% Gel
- (25 mL TBE, 0.25g agarose)
- Incubate gel in ethidium bromide solution for 30 minutes
- Incubate gel in rinse solution for 30 minutes
- Look for band on illuminator
- If band exists, cut out of gel with razor
- Follow protocol from Promega Wizard miniprep kit
- melt the gel for 5-10' at 70°C
- add 1mL purification resin and mix
- put the resin in the column
- wash with 2mL 80% isopropanol
- spin 2' at 14000 rpm
- air dry for 5'
- add 50uL autoclaved water and incubate 2'
- spin 30 seconds at 14000 rpm
Restriction of PCR product
If you had a bright DNA band after PCR, use 20μL of the product. If you had a faint band, lyophilize the 50μL from the previous step and add 20μL water. The restriction enzymes that I need for pQE-80 are: BamHI and HindIII
- Start protocol first thing in the morning
- Do the restriction in a total 50μL mix
- normally choose the buffer that is good for both enzymes, but NEB says to do them sequentially
- Add 5μL HindIII buffer
- Add 24μL water
- Add 1μL HindIII
- Incubate for day at 37
- Add 1μL of 5M NaCl to buffer see discussion thread
- Add 1μL BamHI
- Incubate overnight
- Heat inactivate 20' at 65°C
- (Don't know if I need to gel purify here)
Restriction of vector
Run the cuts at the end of the day
- cut 10μg vector in a 50μL mix
- Add appropriate volume of DNA (check concentration)
- Add 5μL HindIII buffer
- Add appropriate volume of water to 49μL
- Add 1μL HindIII
- Incubate at 37°C overnight
- Run a 1% Gel
- (25 mL TBE, 0.25g agarose)
- Incubate gel in ethidium bromide solution for 30 minutes
- Incubate gel in rinse solution for 30 minutes
- Look for band on illuminator
- If band exists, cut out of gel with razor
- Follow protocol from Promega Wizard miniprep kit
- melt the gel for 5-10' at 70°C
- add 1mL purification resin and mix
- put the resin in the column
- wash with 2mL 80% isopropanol
- spin 2' at 14000 rpm
- air dry for 5'
- add 50uL autoclaved water and incubate 2'
- spin 30 seconds at 14000 rpm
- Lyophilize and resuspend in 20μL water
- perform second cut on vector in a 50μL mix
- Use 20μL from previous gel purification
- Add 5μL BamHI buffer
- Add 24μL water
- Add 1μL BamHI
- Incubate at 37°C overnight
- Run a 1% Gel
- (25 mL TBE, 0.25g agarose)
- Incubate gel in ethidium bromide solution for 30 minutes
- Incubate gel in rinse solution for 30 minutes
- Look for band on illuminator
- If band exists, cut out of gel with razor
- Follow protocol from Promega Wizard miniprep kit
- melt the gel for 5-10' at 70°C
- add 1mL purification resin and mix
- put the resin in the column
- wash with 2mL 80% isopropanol
- spin 2' at 14000 rpm
- air dry for 5'
- add 50uL autoclaved water and incubate 2'
- spin 30 seconds at 14000 rpm
Ligation
This also runs overnight
- Premix vector insert and Mg2+
- 1μL cut vector
- 5μL insert
- 1μL Mg2+ (20x)
- 5μL H2O
- Incubate mix for 5' at 37°C
- Add 2μL 10x ATP/DTT (10mM/100mM)
- Add 1μL T4 DNA ligase
- Incubate 2 hours at RT and then ON at 16°C
Transform
Protocol uses electrocompetent cells. May have to see about using these
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