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cloning a large insert


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5 replies to this topic

#1 janani

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Posted 17 February 2011 - 08:01 PM

hi all,
i am trying to clone a 7kb gene into a 6.2kb vector. I have tried different competent cell strains with no success. I also ran the ligation mix in an agarose gel and saw that ligation is working. How do i proceed with transforming this?
plss help. I am at my wit's end.

#2 scolix

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Posted 19 February 2011 - 08:55 AM

one is the competent cell efficiency, other is if you are using the right antibiotic resistance plates. I am sure you would haver checked these. I would try transformation with a commercial competent cell.

#3 janani

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Posted 15 March 2011 - 02:35 AM

i tried electroporation as well, i get plasmids of size 9-10kb and random sizes of vector and insert when i restrict the plasmid. i always get colonies and they always have plasmids but never the correct size. where am i going wrong?

#4 pito

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Posted 15 March 2011 - 02:45 AM

And you are sure your plamid doesnt contain multiple restriction sites?
And if so: you kept that in mind making the calculations?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#5 janani

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Posted 18 March 2011 - 08:12 PM

ya, i checked for multiple sites in my plasmid, but there are none. i am sure i havent made an error in choosing the sites. i checked and asked a lot of others also to verify :(

#6 pDNA

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Posted 19 March 2011 - 05:42 AM

is it a blunt or a sticky ligation? ...maybe you can give more details?

Regards,
p




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