Problem in picking single colony - (Jul/22/2011 )
I am currently cloning an leukimia gene which has its wild type allele and two mutated types which are both of larger base pair in size.
And i have recieved a blood sample which have all the above three alleles.Then i use its DNA to do a PCR and have confirmed the above three alleles.
Then I use the PCR product to do a ligation process with pGEMt vector.After that, i transform the vector into DH5alpha positive competent cells Then i use the DH5alpha positive competent cells to spread plate.
Afterwards, i pick single colony from the plate and i am confident that i have picked one colony each time only since there were toofew colonies for me to make a mistake.
After that, I put the colony in LB medium and shake overnight at 37 degree.
After one day, i directly use the LB medium with those competent cells to do a PCR since i belive that 94 degree in the denaturation process can directly break the cell membrane of those cells and release the DNA. Thus i can skip the miniprep process.
Unluckily, i have found all the three bands shown up in a single PCR product. And i think that is impossible since a single competent cells could only pick up one vector and that vector should have only contained one allele each. Thus a single colony, after a pcr, should have shown only one of the three allels as mentioned above. And it isvery frustrating for me to get all the three alleles together in one single colony.
Pls anyone help me!thX!!
To estimate the frequency of colonies with the correct insert, 10 無 samples from 10 to 20 cultures can be PCR amplified according to a procedure for amplification of bacterial colonies (D. Gussow and T. Clackson, Nucleic Acids Res. 17 4000 (1989)), using the forward and reverse sequencing primers. 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 無 reaction (0.25 pmol/無) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/無) of excess insert remain in the ligation reaction. 2 無 of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/無). If 100 無 (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 無 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.
Do u mean that i have added excess DNA insert in the ligation process? But i am using TA cloning with the use of pGemt vector, i wonder whether it is possible for two or even three inserts to be ligated onto the same vector at the same time. Taq polymerase will only add a 3'dA on the 3' end of the inserts, and i wonder how more than one inserts can be joined together onto the same vector.
As i am still an undergrad, i dun know whether my above concept is rite. anyway, thx for your detailed reply