15 replies to this topic

[shashababy]

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?

[pcrman]

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?

[shashababy]

pcrman, on Jun 19 2009, 07:05 PM, said:

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?

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

[pcrman]

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.

[shashababy]

pcrman, on Jun 20 2009, 09:14 PM, said:

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.

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。

[jiajia1987]

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.

[shashababy]

jiajia1987, on Jun 24 2009, 01:16 AM, said:

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.

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

[jiajia1987]

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.

[shashababy]

jiajia1987, on Jun 24 2009, 05:19 PM, said:

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.

The lab where the amazing happening !
thanks  pcrman and jiajia1987. both of you are nice people.

[phage434]

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.

[jiajia1987]

phage434, on Jun 29 2009, 04:36 AM, said:

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.

Hmm.. this is interesting. I didnt know that and thanks a lot for pointing this out.

For all the sequences that gave the wrong identity, they had the wrong sequences inserted too. So the problem now here lies with the fact that other things ligated to the TOPO TA vector, rather than the actual insert I want to ligate. And I have no idea where these things came from.

[shashababy]

hi everyone
my insert exsiting recombination which are confirm by other lab.....the copy of iron channel-rRat-Nav1.3  in Ecoli. is more compliacted than we think.

[hanming86]

jiajia1987, on Jun 28 2009, 06:11 PM, said:

phage434, on Jun 29 2009, 04:36 AM, said:

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.

Hmm.. this is interesting. I didnt know that and thanks a lot for pointing this out.

For all the sequences that gave the wrong identity, they had the wrong sequences inserted too. So the problem now here lies with the fact that other things ligated to the TOPO TA vector, rather than the actual insert I want to ligate. And I have no idea where these things came from.

The mus musculus DNA or whatever is really suspicious as phage pointed out. i got it before from a failed direct genomic DNA sequencing. Don't know what to make out from this mus DNA.

shashababy, on Jul 20 2009, 01:41 AM, said:

hi everyone
my insert exsiting recombination which are confirm by other lab.....the copy of iron channel-rRat-Nav1.3  in Ecoli. is more compliacted than we think.

TOP10 is supposed to be recA- i wonder what could be causing this recombination.

[novagen]

Hi everyone

Even I have faced similar problem and tried to confirm by amplifying the cloned product using gene specific primers.
It got amplified very well.

1.Suppose if the clone was a weird band.How do my primers(gene specific primers ) anneal to  the insert.
2. The sequence showed 100% similarity to the one of the phage genes and 98% similarity to one of the plant gene.

ThanX

[jiajia1987]

novagen, on Jul 23 2009, 12:07 AM, said:

Hi everyone

Even I have faced similar problem and tried to confirm by amplifying the cloned product using gene specific primers.
It got amplified very well.

1.Suppose if the clone was a weird band.How do my primers(gene specific primers ) anneal to  the insert.
2. The sequence showed 100% similarity to the one of the phage genes and 98% similarity to one of the plant gene.

ThanX

That have been on my mind for some time and i'm waiting for an answer to it too. lol.
I guess... we have to be skeptical about sequencing results at times.