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Red/ET recombination


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#1 christinedong

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Posted 05 October 2010 - 06:54 PM

OMG, I'm really freaking out about this stupid recombination. OK, here's the question. I have a BAC containing the whole virus genome, and I used Kan and Amp, knocked out two copies of genes. The same gene, just two copies in different regions in inverted repeats. Now, I'm using the same recombination method, try to knock in another gene. Because most of the sequences in inverted repeats are the same, the BAC tends to recombine itself, generating a huge deletion with the whole sequences between Kan and Amp deleted. The PCR product containing my gene of interest, will end up with either digested by endonuclease, or hard to detected because the lack of negative selection. I screened hundreds of hundreds of colonies, most of which are having the huge deletion. I feel like if I'm not lucky enough, I will just spend my whole life screening, but getting nothing.

Does anybody have done this before? Or if there's selection that I can reduce the number of colonies for screening?

I spend almost 1 year to knock out the gene, and don't wanna spend another two just knock in one gene...


Thx!!

#2 pDNA

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Posted 06 October 2010 - 01:02 PM

first of all you can use different E. coli cells to prevent instability due to recombination, like:

STBL2 (Invitrogen)

F- endA1 glnV44 thi-1 recA1 gyrA96 relA1 Δ(lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ-

* host for unstable sequences such as retroviral sequences and direct repeats
* nalidixic acid resistant
* References: Trinh, T., Jessee, J., Bloom, F.R., and Hirsch, V. (1994) FOCUS 16, 78.

STBL3 (Invitrogen)

F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1

* Streptomycin resistant
* endA+, use care in preparing DNA from this strain

STBL4

endA1 glnV44 thi-1 recA1 gyrA96 relA1 Δ(lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ- gal F'[ proAB+ lacIq lacZΔM15 Tn10]

* Tetracycline resistant (from Tn10 insertion)
* STBL2 + blue/white selection

SURE (Stratagene)

endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 uvrC e14- Δ(mcrCB-hsdSMR-mrr)171 F'[ proAB+ lacIq lacZΔM15 Tn10]

* uncertain status of TraD36 in F plasmid
* increased stability for inverted repeats and Z-DNA
* nalidixic acid resistant
* kanamycin resistant
* tetracycline resistant

SURE2 (Stratagene)

endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 uvrC e14- Δ(mcrCB-hsdSMR-mrr)171 F'[ proAB+ lacIq lacZΔM15 Tn10 Amy CmR]

* increased stability for inverted repeats and Z-DNA
* nalidixic acid resistant
* kanamycin resistant
* tetracycline resistant
* chloramphenicol resistant for < 40 μg/ml, sensitive for > 100 μg/ml

The other part of your story i did not really get! Maybe you can explain it in more detail.
You already knocked out two genes using AMP and KAN, right? ...now you want to add a new gene without using a selection marker, right?

My first question is why? ...is there any special reason why you did not add a resistance gene with your gene of interest? ...if so, please explain it! Maybe there is a work-around for your problem!

Regards,
p


OMG, I'm really freaking out about this stupid recombination. OK, here's the question. I have a BAC containing the whole virus genome, and I used Kan and Amp, knocked out two copies of genes. The same gene, just two copies in different regions in inverted repeats. Now, I'm using the same recombination method, try to knock in another gene. Because most of the sequences in inverted repeats are the same, the BAC tends to recombine itself, generating a huge deletion with the whole sequences between Kan and Amp deleted. The PCR product containing my gene of interest, will end up with either digested by endonuclease, or hard to detected because the lack of negative selection. I screened hundreds of hundreds of colonies, most of which are having the huge deletion. I feel like if I'm not lucky enough, I will just spend my whole life screening, but getting nothing.

Does anybody have done this before? Or if there's selection that I can reduce the number of colonies for screening?

I spend almost 1 year to knock out the gene, and don't wanna spend another two just knock in one gene...


Thx!!



#3 donny

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Posted 09 October 2010 - 07:58 PM

There's another protocol that's an extension of the Wanner method that uses 3-step PCR instead of one to generate a PCR product that flanks the gene with 500bp instead of just 50bp. That way, you may get more specificity and be less susceptible to the effects of endonuclease.




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