PCR Screening of recombinant DNA from bacterial colonies
Materials
Transformed E. coli colonies on agar plate(s)
Lysing buffer (10 ml):
Components
Final conc.
Stock
Quantity
Tris-HCl, pH8.5
20 mM
2 M
100 ul
Triton X-100
1%
10%
1 ml
EDTA
2 mM
0.5 M
40 ul
H2O
8.86 ml
Mix, sterilize by 0.2um filter.
96-well PCR plate
Pipette
Displacement pipette and tips
Thermomixer with 96-well plate adapter
Centrifuge
Permanent marker
Ice
Reagents and equipments for PCR reaction
Procedure
Alocate 50ul lysing buffer each well into 96-well PCR plate.
Using displacement pipette inoculate single colony numbered on agar plate, in lysing buffer by repeated pipetting.
(Use a "vector only" colony as the negtive control and target-containing plasmid colony as positive control!)
Write down the numbers on PCR plate corespondent to the colony numbers on agar plate.
Or alternitively making miniculture first then do the following:
Inoculate single bacterial colony in 100 µl bacterial growth medium containing appropriate antibiotic on 96-well round bottom plate
Grow at 37o at 800 RPM using microplate attachment for Thermomixer R ( 2hr to overnight).
Transfer 50 µl to Eppendorf (ED) conical bottom PCR plate well. Pellet in table-top centrifuge 10’ at RT (at 4000rpm).
Remove medium by aspiration.
Lyse bacterial pellet by adding 50 µl lysis buffer (see PCR screening protocol) and shaking at 1400 RPM 15’ in Thermomixer R at RT.
Continue to the regular procedures.
Mix 5min in Thermomixer at room temperature.
Heat at 95oC and shake for 10-15 min.
Transfer onto ice.
Clarify by spining 15min at top speed (4000-14000rpm?) at 4oC
Prepare master PCR mix using standard PCR conditions.
Inoculate 5ul supernatant in 100ul PCR reaction on ice. Plus the following controls:
Negtive control: Master mix + 5ul lysate of non-recombinant colony
Positive control: Master mix + 5ul lysate of known recombinant colony, if any
Empty control: Master mix without template