Help for T-A cloning - (Jun/19/2009 )
HI everyone:
1.I can get 5kb by PCR from a full-legth Nav1.3 (one kind of sodium channels ). I use pfu polymerase from stratagene.
2.Afer PCR , i puried the product using a DNA spin columns ， and then tailing it A for TA cloning .
3.After tailing A, i run a electrophoresis , and extracting the 5kb from agarose.
4. finally, i use the product from the step3 ligation to pMD18-t simple.
5.transformation to top10 Bacteria
the question is here:
when i did Colony PCR from the white spot by primer M13-47 and RV-M, i could get a 500bps, not 5000bps!
The inserts should be 5000bps,because i extracting it from agarose. there was no reason i could get a 500bps inserts.
i extracted the plasmid and did a double-digest， and no dna fragments were cut. why?
Colony PCR results tend to be not reliable for clone identification. Have you run a gel with the original plasmid (thus lineralized) and your cloned plasmid? If there is a 5kb insert, you should be able to see a difference in migration despite your plasmid is circular. To make an accurate comparision, you can cut your plasmid with just one enzyme. Is the double digestion done with two enzymes, if yes, are they compatible with the buffer you used?
pcrman on Jun 19 2009, 07:05 PM said:
i use the fastdigest enzymes from fermentas. and the buffer are only one kind for all the fastdigest enzymes.
one result of my colony PCR is about 1200bps, and i do a double digest with the same plasmid , the result of double digest is also 1200 bps. it is too strange! how could it happened? there is no reason i can got a 1200bps dna fragments from extracting dna from agarose, and the 1200bps dna fragments is aslo having two enzymes sites. i send the strange plasmid to company for sequencing it . it's awful terrible!!h
How many clones have screened? Most likely that you got non-specific amplicon which was somehow not separated well with your 5kb band (I assume that you ran the gel, cut the band and did purification). When you run the gel, make a long gel to get good separation of your desired band from unspecific band. You can also screen more colonies to try your luck.
pcrman on Jun 20 2009, 09:14 PM said:
One result of sequencing is unaccountability. According to the result， i find that there is a 501 bps insert in T-vector. the first 187 bps of this insert is Identities = 187/187 (100%) to some kinds of Ecoli. But the rest of this insert is Identities = 303/313 (96%) to Mus musculus chromosome UNK clone CH35-73N5, complete sequence. So i must say .........it is weird.
I decide to Sequencing my PCR product extracted from the agarose。if the pcr product is what my target is , i really don't know how could this happend...............
there is no mistake during my experiment. Because my postive control is work very well。
My supervisor is kinda skeptical about gel extraction at times. Though we are supposed to get only the fragment we want when we cut it out, the gel tank can contain many other smaller DNA fragments from previous gel electrophoresis or previous gel extraction. This can be significant when it comes to TOPO TA cloning. What he recommended for me to do was to full my gel tank with distilled water, pour a bit of virkon into it and leave it to soak for 1 hour. After which, the gel tank will be washed and left to UV exposure for 5 mins. This step ensures any residual DNA from previous usage to be "killed" as they can just migrate into the gel randomly and get cut out along with the band you want to purify. After all, kits are never 100% reliable.
I had the same problem as you before. I cut out the PCr products that I wanted and ligated it to TA vector, which gave me white colonies, but had the wrong inserts. SOme of the inserts had a short sequence similar to my PCR products while others were E.coli or from mouse genome. Run your PCR products at 80V in a 0.8% agarose gel for 2 to 3 hours. Try running until the band you want is about 1inch above the bottom of the gel and cut it out.
jiajia1987 on Jun 24 2009, 01:16 AM said:
I had the same problem as you before. I cut out the PCr products that I wanted and ligated it to TA vector, which gave me white colonies, but had the wrong inserts. SOme of the inserts had a short sequence similar to my PCR products while others were E.coli or from mouse genome. Run your PCR products at 80V in a 0.8% agarose gel for 2 to 3 hours. Try running until the band you want is about 1inch above the bottom of the gel and cut it out.
hey ,body.you are awesome! But can u tell me where the product which contain E.coli genome and mouse genome come from? In my lab ,there is no one research the genome of both Ecoli and mouse. can u tell me your msn address? thanks alot
Honestly, I have no idea where the mouse genome and all those stuffs came from!
In my lab, no one was working on all these stuffs. The only thing about the E.coli genome could be because of contamination since molecular cloning usually makes use of E.coli. I once had a sequence that had some bit of homology to flavivirus (the dengue family) and HIV, but NO ONE was working on it. Even my supervisor was puzzled.
jiajia1987 on Jun 24 2009, 05:19 PM said:
In my lab, no one was working on all these stuffs. The only thing about the E.coli genome could be because of contamination since molecular cloning usually makes use of E.coli. I once had a sequence that had some bit of homology to flavivirus (the dengue family) and HIV, but NO ONE was working on it. Even my supervisor was puzzled.
The lab where the amazing happening !
thanks pcrman and jiajia1987. both of you are nice people.
The contamination in many of these cases is in Genbank, rather than in your lab. The quality and identity of many Genbank entries is suspect. Many cases of vector contamination and E. coli genomic DNA occur and are mis-labeled in the database. You can usually believe the Ref-Sequence data, but the other Genbank data should be taken with a few grains of salt.