colony PCR screening, drove my crazy! need ur suggestion - (Aug/21/2007 )
i use TOPO-TA cloning kit from invitrogen to clone different size purified DNA into vector.
sample DNA 1: 1050bp
sample DNA 2: 600bp
sample DNA 3: 300bp
sample DNAs 4: 650bp, 450bp (mixture, cause the protocol hints that it could also clone PCR pool, meaning DNA mixture?)
after LB+amp culture, i got hundreds of colonies for each sample.
firstly, i use colony PCR to verify the insert size for sample DNA1 (1050bp) with primer M13(RE) M13(FOR), and it turns out 1100bp size.
But i'm wondering if it shoud be larger than original 1050bp? cause there are almost 130bp between M13 primers and PCR product insert site on the vector.
then, i colony pcr sample DNA2, sample DNA3 with primers T3, T7. The 20 colonies of sample DNA2 (600bp) give ALL same size band, 750bp! For sample DNA3 (300bp), the bright bands are also of around 750bp! and most of them have smear around 200-300bp.
The question is why they all have the same size 750bp very bright band?
Are those smear the right insert but of high colony input?
last, sample DNA4, mixtures (450+650bp), out of 10colonies, 5 colonies have extreme large size band, >3000bp, and also smear around 400-500bp.
what are those large size band? the vector sequence?
again the smear indicate the right insert size?
i don't know how to explain these result, and time is passing. i have no more than one month to get all results.
Should I colony PCR them again with lower concentration of colony suspension, hoping to solve the smear problem?
Or use restriction enzyme digestion instead of colony PCR?
Thanks in advance!
If the colony PCRs are giving you trouble and you don't have much time to get the results, just go to restriction digests. Much simpler!
sample DNA 1: 1050bp
sample DNA 2: 600bp
sample DNA 3: 300bp
sample DNAs 4: 650bp, 450bp (mixture, cause the protocol hints that it could also clone PCR pool, meaning DNA mixture?)
after LB+amp culture, i got hundreds of colonies for each sample.
firstly, i use colony PCR to verify the insert size for sample DNA1 (1050bp) with primer M13(RE) M13(FOR), and it turns out 1100bp size.
But i'm wondering if it shoud be larger than original 1050bp? cause there are almost 130bp between M13 primers and PCR product insert site on the vector.
Yes, the PCR product should be larger, after all the M13 primers are actually binding just beyond the peice of DNA you have added.
The question is why they all have the same size 750bp very bright band?
Are those smear the right insert but of high colony input?
last, sample DNA4, mixtures (450+650bp), out of 10colonies, 5 colonies have extreme large size band, >3000bp, and also smear around 400-500bp.
what are those large size band? the vector sequence?
again the smear indicate the right insert size?
Large band sizes, are probably random bits of genomic DNA being amplified. If you can show the gel picture it would be really helpful. As you have suspected, it is very possible that you are using too much cells. Could you write down what formulation you are using? I myself, pick one medium size colony(16hr growth) and dip it into 50ul of sd water. Of that I use only 2.5ul and add that to the PCR mixture (giving a final volume of 10ul) Number of cycles is 33. Extention time is 1min at 72 Celsius. M13 primers is I am not mistaken use 50 Celsius for 30 Sec. (but do check, my M13 primers may not be the same as yours)
Or use restriction enzyme digestion instead of colony PCR?
Hard choice, but trial and error I have become very confident with my PCR results. If it says it isn't there, painful as it is, it isn't there. I am not sure how confident you are with the colony PCR technique. How about this, try once more with colony PCR (with one plasmid type), dilute the cells. If that doesn't work, use the miniprep.