update of colony-PCR result --screening for correct recombinants from 4-way liga - (Oct/13/2006 )
Hi,
I am writing to report the colony-PCR result.
before performing colony-PCR, I first streaked the colonies onto fresh plates to get single colony and at the same hoped to "dilute" any DNA contamination (if there were). Then yesterday I picked the colony, streaked onto another plate to make a stock and resuspended the residual cells into 50ul water.
I tried colony-PCR on 60 ramdonly picked colonies that seem to be resistant to both Cm and Erm (as seen from plate streaking result). The targeting sites for the two primers I used: one is vector specific (targeting site is on the backbone) and the other one is targeting the 1st junction where ErmR cassette (2nd piece) ligated with the first piece. And out of these 60 colonies screened, about 45 yielded positive results, meaning there is a product of 1.5kb which was expected.--but isn't the effiency of seemingly-correct ligation too high?
Then today, I happily picked 30 out of these 45 (by scratching the stock streaks I patched yesterday) for the second colony-PCR screening (using another primer pair that target the 2nd junction site and the other side of vector backbone)--but I got no positives for any of the 45 colonies screened....
WHY???--I setup the exactly the same PCR reaction (of course using different primer pair this time) except that I may scratch too many cells out of the colony to make template suspension in the 2nd colony-PCR. I noticed that the 50ul of suspension seemed rather thick and even some cell debrie were floating around!!! If this is the proble, I would not worry too much...
But the thing I am worrying is--is possible that the results from 1st colony-PCR may have mislead me? Is it possible for any false-positive to arise? By streaking 3-4 times on both Erm and Cm plates, I am pretty sure the ones picked are resistant to both antibiotics. And if there were no misleading false-positives from 1st PCR, in theory, the ones picked should yield positive signal in the 2nd PCR. Right? But if there were false-positives from 1st PCR, how did it happen?
Tomorrow, I will try another colony-PCRs: I will run 3 PCRs simultaneously for each colony-picked: the primers targeting 1st junction, 2nd junction and ErmR cassette. If any one shows positive in all the three reactions, maybe it carries the true recombinant plasmis desired.
And of course, I will be careful not to scratch too many cells next time...
any comments and tips to make improvement?
It could be your template or that the PCR conditions will need to be optimized for the second set of primers...
However, since you had such positive results with your first PCR, I would mini prep a few of the positive ones and then check by restriction digests. At the very least this will tell you if your first PCR result was real, and hopefully you can also identify one with the 4 correct inserts.
I am writing to report the colony-PCR result.
before performing colony-PCR, I first streaked the colonies onto fresh plates to get single colony and at the same hoped to "dilute" any DNA contamination (if there were). Then yesterday I picked the colony, streaked onto another plate to make a stock and resuspended the residual cells into 50ul water.
I tried colony-PCR on 60 ramdonly picked colonies that seem to be resistant to both Cm and Erm (as seen from plate streaking result). The targeting sites for the two primers I used: one is vector specific (targeting site is on the backbone) and the other one is targeting the 1st junction where ErmR cassette (2nd piece) ligated with the first piece. And out of these 60 colonies screened, about 45 yielded positive results, meaning there is a product of 1.5kb which was expected.--but isn't the effiency of seemingly-correct ligation too high?
No. The presence of the 2nd (ErmR cassette) is ~100%, you should get nearly 100%.
WHY???--I setup the exactly the same PCR reaction (of course using different primer pair this time) except that I may scratch too many cells out of the colony to make template suspension in the 2nd colony-PCR. I noticed that the 50ul of suspension seemed rather thick and even some cell debrie were floating around!!! If this is the proble, I would not worry too much...
That would certainly be a major cause of PCR failure.. The cell suspension at most should be only slightly cloudy. It actually should look frighfully clear (like there is nothing much in there). trust PCR to amply your signal. I use 34 cycles...
How much colony solution are you using?
If face with this situation again, I would use dilute the single colony in 100ul and use maybe 2ul. Don't drop the volume added to the PCR mix, it becomes harder to accurately pipette 1ul compared to 2ul. Just dilute the colony solution more.
PCR done without contamination doesn't lie... just answers a very restricted meaning of the truth. It can mislead in the way that 1st junction (Emr cassette is ligated to the vector) is present, but no where does it say that the 2nd junction (insert 2 ligated to vector) is present.
And of course, I will be careful not to scratch too many cells next time...
any comments and tips to make improvement?
Just make sure your cell suspension isn't too thick or your cells too old (keep them refrigerated if not in use).
Hi, good morning...thanks for the tips and comments
this morning, I set up colony-PCR again--and I paid extra attentio to make sure not too many cells were suspended. At least, this time I can say the suspension is clear....And the colonies were fresh overnight-streaks grown on BHI+Erm plate.
And as usual, I resuspended the cells in 50ul of Rnase-free water, breifly votexed the suspension and use 2ul for PCR. The final volume of PCR mix is 10ul (8ul mix+2ul suspension). And I increased the cycles to 34 as you recommended. .. Two sets of PCR were set up for each colony picked, hoping to amply out the 1st insert and 2nd insert. Since ErmR cassette is there and expresses so well, I did not bother run PCR using ErmR-specific pair of primers.
OK, I will keep you informed of the results later this afternoon...
cheers,
Paula