Protocols
LB medium
Materials
10g of tryptone
5g of yeast extract
10g of NaCl
top up to 1L (with deionised water)
Procedure
1.Use a container a container with at least double the volume of the LB that you are making.
2.Measure out the weights of tryptone, yeast extract and sodium cholride as above then fill up with deionised water to 1l and mix well until clear.
3.Ensure the lid is unscrewed by two and a half turns
4.Send to be autoclaved
LA medium
Materials
10g of tryptone
5g of yeast extract
10g of NaCl
15g of agar
top up to 1l (with deionised water)
Procedure
1.Use a container a container with at least double the volume of the LA that you are making.
2.Measure out the weights of tryptone, yeast extract, sodium chloride and agar with deionised water to 1l and mix well.
3.Ensure the lid is unscrewed by two and a half turns.
4.Send to be autoclaved
5.Pour the plates next to a Bunsen burner
6.Leave for 15-20 minutes to set/solidify
Mini prep
Harvest bacterial cells
Pelet 20ml of saturated E. coli for 60 seconds at 11,000 x g.
Discard supernatant and remove as much liquid as possible
Lyse cells
Add 500ml Resuspension Buffer P1 and resuspend cell pellet by vortexing.
Split the solution into two 1.5ml microcentrifuge tubes.
Add 250μl Lysis Buffer 2
Mix gently by inverting tube 8 times,
Incubate at room temperature for five minutes or until lysate appears clear.
Add 300μl Neutralization Buffer 3.
Mix thoroughly by inverting tube 8 times.
Clarification of lysate
Centrifuge for five minutes at 11,000 x g at room temperature
Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50օC
Bind DNA
Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube
Pipette a maximum of 750μl of clarified sample supernatant onto column
Incubate at frrom temperature for two minutes.
Centrifuge for one minute at 11,000 x g and discard flow-through.
Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarifed sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.
Wash silica membrane
Add 500μl Wash Buffer Pw1
Centrifuge for one minute at 11,000 x g
Add 600μl Wash Buffer PW2 (supplemented with ethanol)
Centrifuge for one minte at 11,000 x g
Discard flow-through and reuse Collection Tube
Dry silica membrane
Centrifuge for two minutes at 11,000 x g, to remove residual ethanol
Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.
Elute DNA
Add 50μl Elution Buffer P directly on the top of the silicon matrix
Incubate at room temperature for two minutes
Centrifuge for one minute at 11,000 x g.
Gel extraction
Excise and dissolve gel slice
Using a clean sclapel excise DNA fragment from gel
Remove excess agarose, determine weight of gel slice and transfer into a clean tube
Add 200μl Binding Buffer CB per 100mg of 2% agarose gel
Incubate sample at 50օC for ten minutes, vortexing sample briefly every three minutes until gel slice is completely dissovled
Incubate at room temperature for two mintues
Bind DNA
Place ISOLATE II PCR and Gel Column in a 2ml Collection Tube and load 600μl of the sample
Centrifuge for thirty seconds at 11,000 x g and discard flow-through
Reuse collection tube for step 3
Wash silica membrane
Add 700μl Wash Bufer CW to ISLOATE II PCR and Gel Column
Centrifuge for thirty seconds at 11,000 x g
Discard flow-through and place column back into collection tube
Repeat step three to minimize chaotropic salt carry-over
Dry silica membrane
Centrifuge for one minute at 11,000 x g, to remove residual ethanol
Place ISOLATE II PCR and Gel Column in a 1.5ml microcentrifuge tube
Elute DNA
Incubate at room temperature for three minutes
Add 15-30μl Elution Buffer C directly onto silica membrane
Incubate at room temperatre for three minutes
Centrifuge for one minute at 11,000 x g.
"
