Hi,
I am quite new in cloning and have been trying very hard to troubleshoot cloning problems that I'm currently having. I am trying to create gene clone libraries for assA gene using pGEMt cloning kit.
I have amplified the gene (produced right size with no unspecific amplification), purified the amplicons (Qiagen kit) and prepare 8:1 ratio of amplicon:vector. I prepare the ligation reaction according to the procedure and incubate overnight at 4 degree. Then I transformed the plasmid into DH5a competent cells and grow them over night in gel with amplicillin, IPTG and Xgal and I get good ratio of white to blue colonies. Positive controls have 70-80% white colonies (100 colonies), background control only have 3 blue colonies and the transformation control with pUC19 plasmid were all blue colonies (~120 colonies).So far all is good I think?
I pick the individual white colonies and grow them on LB media with ampicillin (~3ml each colony) overnight and did both: 1) colony PCR with insert specific primers and 2) miniprep and sequencing. I don't get the right band size for the colony PCR and the random plasmid that I sent for sequencing did not contain the insert. My question is why don't I find my insert in the white colonies and what can I do to fix this problem?
I'm already at my limit now. I'm like an active volcano waiting to erupt! Your wise suggestions will greatly be appreciated!
The fragment is 550 bp. What do you mean by multiple of three?
If your fragment was 333 bp, it is evenly divided by 3 and 3 nucleotides makes up a codon/amino acid. Your fragment is actually 552 bp with the A overhang on each end which is a multiple of three. I made a typo above when I said "it might be interrupting the lacZ gene which means your clone might be in the blue colonies". It should be "it might NOT be interrupting the lacZ gene...".
You might want to check a few of the blue colonies and see if your insert is there.
Good Luck!
Hi There,
Try to do a colony PCR using primers on the vector that flank your insert. If the PCR product has about the same size of the amplicon you got with the insert specific primers, then sequence two of them. This may give a clue of what is happening to your insert.
Alternatively, your insert may not be stable and has been modified after transformation.
Lastly selecting one plasmid randomly without being sure if it has the insert was not a good idea.
Hope you will find it helpful.
hi!
Better to do a Pcr to check ur possitive clones, I will recomend u to extract the DNA for some possitive colonies and make a diggestion that allow u to see ur insert and ur vector! As Tom suggest, pick also some blue colonies to be sure u are cloning in the proper possition. Colony PCR is not always working for any reason, so to be 100_% that u do not have ur clon check by diggestion.
Hope I help a bit!
Can you provide the sequence result you obtained? Seeing what kind of scar, if any, is present may help get a better answer
Tom Masi: If I don't even have my insert in the white colonies, why should I bother check insert in blue colonies?
Devin: The sequence has high similarity to vector pGem that I'm using. All of them!
Now I think it's possible that the polymerase in the master mix (Accustart II PCR tough Mix) was not as potent as regular taq polymerase to add A-overhangs to my amplicons thus none of them were ligated to the vector. But that still cannot explain why I get tons of false positive (white colonies but with no insert).
You did a colony PCR with insert specific primers- what were the size (bp) of your product?
You may want to check the blue colonies because your DNA fragment is small and is a multiple of 3 and therefore would not interupt the lacZ gene and you would still have blue colonies. If the DNA fragment was large it would interupt the lacZ gene and the colonies would be white.
I don't think you need to check dark blue colonies with an insert >500bp. In my experience, sometimes very short (e.g. 201 bp) products will result in white colonies with a pale blue center due to some LacZ still being functional despite the short insertion, but the colonies are still mostly white.
Do you have a positive control for your colony PCR to rule out bad PCR reagents? You should try using just your insert as a positive control.
Another option: PCR with plasmid-specific primers. Use a blue colony as a negative control. It should result in a short band. Your white colonies should result in a higher MW band.
Another thing to consider: What % GC does your gene contain? If it is >60%, your colony PCR might be failing due to poor amplification. You could try miniprepping a few of the white colonies and doing a miniprep PCR instead. That is more likely to work since your template is more concentrated and pure.
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