screening recombinant colonies - (May/30/2007 )
hi to all of you there
i just read in one of promga's notes that to determine the percentage of true recombinants, rapidly perform a screen by picking a number of white colonies and resuspending each one in 50μl of sterile water. Boil for 5-10 minutes and spin in a microcentrifuge for 2-3 minutes to pellet cell debris.
how can i boil the water in the tubes??? and is it ok if i do it 2days after getting the white colonies please forgive me for my stupid question iam very new to the whole thing
any help wll be really appreciated
You can either put it in boiling water or just microwave.
Longer denaturation step during PCR will do the trick. 
what if i just do PCR with my insert's primers to the mini prep plasmid from the white colony is it considered a way o screen the recombinant colonies????
Do not use the both of the insert's primers to check for positive colonies.
Colony PCR initiated from transformation reactions can give rise to false positives if the primers anneal to the insert sequence due to excess DNA insert from the ligation reaction that is spread as part of the transformation reaction onto bacterial plates. (Qing Dallas-Yang, Guogiang Jiang and Frances M. Sladek, 1998. Avoiding false positives in colony PCR. BioTechniques 24 (4) 580-582.) Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL reaction (0.25 pmol/µL) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/µL). If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018 pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 e 10 molecules) of background insert are available for a 100 µL colony PCR assay. To avoid this problem, at least one primer should anneal to the vector. Use of the Forward and Reverse Sequencing primers is recommended, as this allows the determination of double inserts or vector-only colonies. A negative control consisting of vector-only is also recommended for purposes of band identification.
Spinning the boiled cells removes the cell debris which can cause smearing of the DNA.
YOU CAN DO THAT AS WELL BUT COLONY pcr IS QUICK YOU GET THE RESULTS SAME DAY YOU'VE PICKED A COLONY.
FOR BOILING YOU CAN ALSO USE A HEATING (SET AT 100C) OR USE A WATERBATH ALTHOUGH YOU HAVE TO BE CAREFULL