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ligation and transformation problems! Urgent!


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#16 netnus

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Posted 04 December 2004 - 07:03 AM

HI ,
To standardise the ligation reaction one easiest step u can follow is
digest ur plasmid with one of ur enzyme and ligate back  in diffrent conditions
and look for colonies .
perform a transformation with these contols
1) uncut plasmid
2) single cut plamid
3) single cut plamid + ligase
this approch at least will allow to check where is the problem??

bye
praveen

Thanks for your advice! I do try all the steps you say and the single cut plasmid+ligase grows a lot of colonies while single cut plasmid not. The problem is mostly likely the second digestion especially in this case BamH1.
I am going crazy in this step!!


netnus

#17 praveen_iitb

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Posted 05 December 2004 - 08:52 AM

HI,

So at least u come to know that BamH1 is not cutting ur DNA. Did u try the transforamtion with Plamsmid cut with only BamH1. If u observed colony in this step then something serious with plasmid preparation. Or u can do one thing
----First cut ur plasmid with BamH1 and then gel purified the fragment (linear one) to avoid uncut plasmid
----then cut with ur second enzyme. and set up a test ligation.
This step surely will give u some posotive results.
bbye

#18 Janina

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Posted 06 December 2004 - 12:24 AM

I don't think that it is necessary to purify by gel after first digestion. The loss of DNA could be to high... But it is necessary to refresh buffer after first digestion.

#19 netnus

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Posted 06 December 2004 - 07:12 AM

I don't think that it is necessary to purify by gel after first digestion. The loss of DNA could be to high... But it is necessary to refresh buffer after first digestion.

Things are getting better! I finally see some colonies in the plate. I pick all of them and grow in the LB mdium.
After extraction, I run PCR and yes,I can see the pcr product in the right position. Next I would try double digestion to see if I can see the insert band.
Thank you all very much for your generous helps!

netnus

#20 Janina

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Posted 06 December 2004 - 11:49 PM

And what did you change at least?

#21 netnus

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Posted 07 December 2004 - 07:36 AM

And what did you change at least?

I re-double cut the vector in a relative short time 1.5h.
I change the ratio of insert to vector. I usually set up the ligation based on the mass ratio of 3:1. But this time I change the mass ratio to molar ratio. I remember someone tells me that too much insert will inhibit the ligation.

ng of insert=ng of vector*(kb of insert/kb of vector)*(3/1)

Edited by netnus, 07 December 2004 - 07:39 AM.





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