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helena kyle

Member Since 13 Oct 2012
Offline Last Active Dec 17 2012 08:42 PM

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yeast,cloning,expression, knock-out

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helpwithdna
16 Dec 2012 - 15:16

Topics I've Started

cloning pcr insert into plasmid

16 December 2012 - 12:34 AM

Hi,

i am trying to clone a pcr insert of 2kB into a plasmid pYES of 6kB.

i designed the primers (2bp additional nucleotides+NcoI sequence+16bp gene sequence)

the pcr insert is purified,then digested with NcoI (37degC, 3 hours). the plasmid is digested with NcoI too.

after ligation, the ligation mix (10 microL) is electoporated into competent E.coli, all i obtained is many,many,many empty plasmids (without insert)

before, i have tried to dephosphorylate the digested plasmid, but i obtained no colonies on LB ampicillin agar at all

i suspected that it is either the insert is incompletely digested with NcoI or the insert: vector ratio is incorrect (i put 6microL insert:2 microL vector).

any suggestion? is it possible that only adding 2bp additional nucleotides before the RE sequence caused the incomplete digestion?

thanks

colony cracking for screening of positive clones

16 October 2012 - 11:54 PM

Hi,

I have tried the colony cracking method to screen for postive clones. The pYES260 plasmid size is 5933bp.I digested it with BamHI (1 site) and filled-in to create blunt end plasmid. My PCR insert is about 2kB. After electroporation and plating, I extracted the plasmid using colony cracking. However I am not sure which band represented my plasmid.

The first lane on the upper left is the pYES plasmid while other lane are my clones.

Is what I assumed correct?

The pink line is the band for genomic DNA
The blue line is where two bands were observed would be the RNA (Is the faint band across the lane is RNA?)
The purple line is where the proteins were.
The yellow line is the PLASMID.

Another thing is, when I extracted the plasmid, it yielded the three bands. I am not sure of which size should I refer to compare with the clones.

Can anyone have a look at the gel picture attached?

-Helena Kyle-
Thank u Posted Image