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problem in plasmid isolation


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5 replies to this topic

#1 novagen

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Posted 28 February 2010 - 07:13 PM

Hi all,

I have constructed CDNA library from a plant species. every thing appeared right till TA cloning and colony PCR with M13 primers. but no plasmid observed after plasmid isolation . dont understand whats the problem with this since one and half month . hope some one helps me.
1. the competent cells athat I have used are working for other expts.

Thanx a lot in advance
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#2 HomeBrew

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Posted 01 March 2010 - 05:46 AM

Are you performing the colony PCR right from the original transformation plates? We always find an unacceptable number of false PCR positives if we do this. When we first patch colonies to be screened by PCR to a fresh plate, allow them to grow overnight, and perform colony PCR on them (see next sentence), the false-positive situation improves dramatically. Additionally, when we take a small amount of the colony and resuspend it in 50 ul of sterile water and use 1 - 2 ul of this suspension as template for the colony PCR, rather than suspending the colony sample directly into the PCR mix, the false-negative situation is greatly improved as well.

#3 novagen

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Posted 01 March 2010 - 06:28 AM

thankyou for the reply

Actually I have restreaked the colonies from the master plate ,grown over night, nextday resuspended the colony in 100ul of LB broth ,taken 1ul as template for PCR.Iam getting some amplification for colony pCR but when I isolate plasmid from that colony no plasmid bands were obsereved. struggling a lot with this problem.

ThanX
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#4 snoopyx

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Posted 03 March 2010 - 08:34 AM

thankyou for the reply

Actually I have restreaked the colonies from the master plate ,grown over night, nextday resuspended the colony in 100ul of LB broth ,taken 1ul as template for PCR.Iam getting some amplification for colony pCR but when I isolate plasmid from that colony no plasmid bands were obsereved. struggling a lot with this problem.

ThanX


Do you mean no correct plasmid bands were observed, just empty vector? Or you didn't have plasmid DNA at all on your gel? Did you measure the DNA concentration of your miniprep? Is there DNA?
If you have just empty vector on your gel than your ligation didn't work probably. If you don't have any DNA at all than that's a completly different story.

#5 novagen

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Posted 04 March 2010 - 07:57 AM

Hi

No plasmid DNA was observed at all. but if the ligation didnt occur, howcome I am getting PCR amplification (colony PCR) with the M13 primers.

thanx
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#6 hanming86

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Posted 04 March 2010 - 08:14 AM

1. check ur plasmid kit.
2. use higher antibiotic concentration.
3. the m13 primer could be non specific binding
4. kinda unlikely - recombination event ?
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