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plasmid construction where I have to replace the chloramphenicol with kanamycin

Plasmid

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#1 snsjnc

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Posted 15 February 2012 - 03:28 AM

Hi guys. I have to remove the chloramphenicol resistance marker with the kanamycin marker by the restriction digestion.
The CAT(Chloramphenicol acetyl transferase) gene is in donar plasmid(chaperone plasmid) : when I analyze for the unique restriction site in chaperone plasmid, the site is well before(upstream) of the ORF of CAT. The problem I cant use that site since it could be the regulatory region for the CAT gene. I found using the PPP(Prokaryotic Promoter Prediction), it is a promoter
Q1)I need to design the strategy in which I have to replace the CAT gene with KanR gene?

Q2)What I thought is replace the entire set of CAT orf along with its upstream elements and add the separate promoter and RBS(Shine Dalgerno sequence) with kanamycin and replace it. In that context; which promoter(constitutive) and RBS to go for?

I really need suggestions pls.

#2 ThibautEBV

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Posted 15 February 2012 - 04:32 AM

Hello,

You do have only 1 RE possibility? Cause, usually between ORF and promoter you should have MCS, check it again using any DNA software like MacVector.

About your second question. For the promoter, I think look out for classical bacteria promoter used for gene resistance expression (for eukaryotic expression CMV is the best).

Thats not very usefull, sorry.

Good luck

Edited by ThibautEBV, 15 February 2012 - 04:44 AM.


#3 snsjnc

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Posted 15 February 2012 - 04:50 AM

thanks yar. I wan to express that in E.coli yar.

#4 phage434

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Posted 15 February 2012 - 06:09 AM

You can most easily do this by two PCR reactions. Choose an enzyme (or a pair) that are not present in the regions of the parent DNA that will be part of your final construct.
Design PCR primers to the plasmid to amplify the portion of the plasmid without the CAT cassette. Similarly, a pair of primers for the Kan cassette. Add the restriction site(s) chosen to the 5' end of the primers. Add 4-8 bp of extra junk 5' of the restriction sites.

PCR with both sets of primers (independently, or if you are brave, in a single reaction). Mix, digest with the enzyme(s), heat kill, ligate, transform. No purification at any stage necessary.

#5 snsjnc

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Posted 16 February 2012 - 04:02 AM

Any other strategies I can take into consideration yar????

#6 Evanescence

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Posted 17 February 2012 - 05:57 AM

why dont you pcr KanR gene together with the original promoter (if you take it from other plasmid) ??
I would suggest you to use ribosomal promoter of E.coli..again i have to agree with phage434..it's the best way!

#7 phage434

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Posted 17 February 2012 - 03:31 PM

I realized I mis-spoke about no purification being necessary. You must purify the PCR product before the restriction digestions, otherwise the PCR enzymes and dNTPs will extend the cut RE end and make cloning non-functional. Buf that's it.





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