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Help for T-A cloning - (Jun/19/2009 )

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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.

-jiajia1987-

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.

-shashababy-

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.

-hanming86-

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

-novagen-

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.

-jiajia1987-

theres no doubt on sequencing results
bocoz I did restriction site analysis of the clone after getting the clone sequenced.
that was absolutely right
Thanx

-novagen-
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