Using a scfv phage library and doing 4 rounds of biopanning against tumor antigen I infected TG1 cells with the phage from the fourth round of biopanning. Infected cells should then contain the phagemid vector and ampicillin resistance. However, colony PCR with established primers shows that only roughly 1/3 of ampicillin resistant colonies contain the phagemid. The colonies I am picking for PCR are not satellites and grow even on plates with 300ug/ml ampicillin (3x normal). Could this be a result of bad biopanning? Can anybody think of a mechanism by which these colonies could be appearing. The only (longshot) idea that I had was that phage which had mutated to become higher expressors were being amplified by the biopanning but could not be detected by my primers. Any other ideas?
Where do your primers anneal? If they are able produce a visible band even in the absence of scFv insert, theoretically yo should always detect the presence of your phagemid with them. But if they need a scfv gene to produce a visible bad, you can have colonies with phagemid, but without the insert, which is not so rare after panning. This is a likely explanation in my opinion.
Also, sometimes the colony pcr doesnt work well for all the clonies. Did you rule out gross problems affecting colony pcr efficiency?
I would not think in phagemid absence as a likely explanation.
The appearance of mutations precluding amplification doesnt seem very likely, but if it happendene and you have multiple copies of thist mutated clone dominating he sele, your explanation could be correct. If this is the case, you could try to miniprep plasmids from some of his pcr-negative colonies and sequence them. Perhaps you wold find the same sequence, that is not amplified because of some reason.
Thanks for the response. The primers for the colony pcr flank the cloning site and phagemid dna without an insert shows strong bands 700 bp smaller than phamemids with the insert. I think it is unlikely that the colony PCR is just not working for some of the clones because when I do colony pcr on clones from a biopanning that was successful 95% of 20 clones show the expected bands (compared to my
You say that your negative cones have random sequences, but I want to define three things.
1.Do you get plasmids with the expected size of the phagemid from your clones? If so you culd also do a restriction digest with known sites.
2. When you try to sequence these plasmids, can you get a readable sequence using phagemid-specific primers?
3. If so, can you identify phagemid sequences at any position?
Im trying to know wjhether you have evidences of the resence of the phagemid.
Gertrudis,
By random I mean nonreadable, no clear signal and the software I use basically creates a random sequence. So in response to your previous 3 clarifications.
1) No I do not get plasmids of the expected size when I run the mini prep DNA straight out on a gel.
2) No I don't get a readable sequence
3) No I can't identify any phagemid sequence alignments in the sequences from negative clones.
So how are these clones appearing? Its not my plates or technique because when I use a round 4 phage library from other people in my lab I get 95% positive clones.
it appears to be a nice discussion, just to chip in, may be the ampicillin used for the plates is bad, did you tried any experiments to confirm this.
Yes I have. Ampicillin kills 100% of non infected cells. The stock was bought only a week or two ago. And negative colonies will even grow on plates with 300 ug/ml at roughly the same 4:1 ratio of negative to positive. Thanks for the suggestion though.
I have exactly the same issue. Colony PCR shows that more than 80% of them are empty. A few of them give less than expected size. When extract the plasmids and run a gel, they give several abnormally high molecular bands. Using this abnormal DNA as template to do PCR with phagemid as control, it shows no band at all, suggesting this is not phagemid. Cells are OK as culturing these negative clones in LB and re-plating them on LB-CARB vs. LB shows that 99% of them cannot growth. This is result of round 1 biopanning.
Continue to to round 2 and 3, I have more positive clones. I wonder if this is just normal. I wonder if anyone has tried or is willing to try infecting cells with a tiny drop of phage library in your lab to see if you have a lot of colonies without the right phagemid.
Sounds like something happened during library construction. Its normal to have 80% empty after R1 in my experience.
the vector is PADL22c my working hypothesis for this is that bad ampicillin stock allowed for evolution of mutant phagemids.
I have the same issue here. I get expected vector band size after plasmid prep, but i get no band for cPCR using phagemid specific primer. I can’t get a readable sequence using the phagemid specific primer.Aidan Cowan How did you solve this issue?
Dear researchers,
I am having the same problem. After 3 rounds of panning, we isolated positive clones with reasonable monoclonal phage-ELISA signal. However, when we analyzed the phagemid preps from positive clones, we found plasmids with abnormal sizes and without having the antibody sequence in PCR. Anyhow, these clones are able to survive in Ampicillin media. How did you solve this problem?
Aidan Cowan
Michelle Teo
Hi, is there any update from the post after a couple of years? I am curious of how it turns out? Appreciate it if there is any update. Thank you