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Low copy number of plasmid


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3 replies to this topic

#1 Ali

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Posted 27 November 2004 - 07:46 AM

Dear all

I have ligated DNA target (that might be toxic for cell) to pGST-4T-2 plasmid. But, copy number of the recombinant plasmid was very low. Does anyone can help me how to get the enough amount of the plasmid after mini preparation.

Thank ;)

#2 ravibiot

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Posted 27 November 2004 - 08:14 AM

Hi
1. u can try to grow cells in terrific broth
2. check the insert size limitations of the plamid
3. ensure your selction pressure conc. (antibiotic)

ravi

#3 Ali

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Posted 27 November 2004 - 09:08 AM

Dear Ravi

Thank a lot
I cultured plasmid-bearing E. coli in 4 ml of LB medium (with 4 mikroliter of 50 mg Amp). The size of insert is 200 bp.

#4 Janina

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Posted 30 November 2004 - 12:58 AM

Try 10 ml culture! It works most of the time fine... Then elute with warm buffer/water (I use 30 C) - this will increase the yield.




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