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# Procyon

Member Since 31 Jan 2013
Offline Last Active Apr 28 2013 05:28 PM

### In Topic: Problem with ligation using pJET and pGEM for clone library

06 March 2013 - 12:00 PM

bob1, on 28 February 2013 - 12:06 PM, said:

The calculation for molar ratios is:

insert ng = ratio x [insert length /vector length] x vector ng

where the ratio is a number, e.g. for a 3:1 you would use "3".
Insert and vector is length in bp.

This fixed my problems. Thank you very much.

### In Topic: Problem with ligation using pJET and pGEM for clone library

28 February 2013 - 06:20 AM

phage434, on 14 February 2013 - 01:57 PM, said:

If your control reaction transforms 10 ng of DNA, that is 10**-2 ug. If your cells had a transformation efficiency of 10**8 cfu/ug (which is typical for good chemically competent cells) you should get 10**-2 x 10**8 = 10**6 transformants. You are getting 200 instead, so this is your problem. Get cells that 5000 times more competent.

I used new cells and got thousands colonies colonies in the transformation control and got 83 transformant colonies with my insert. 83 is the higest number I have got so far.

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you should limit the ligation to 50 ng or less of plasmid DNA, which given the molar ratios (you do know it is molar, not ng ratio!) of insert:vector will mean a lot less nsert than you are using.

I estimate the ng quantity of my purified PCR product using a 100 bp ladder, and by knowing that the vector is 50ng/ul I estimate the 3:1 ratio of respective ng in the ligation mix. How do I calculate the proper molarity?

### In Topic: Problem with ligation using pJET and pGEM for clone library

14 February 2013 - 01:04 PM

bob1, on 01 February 2013 - 02:53 PM, said:

Ok.  Most of that should be fine, though you should limit the ligation to 50 ng or less of plasmid DNA, which given the molar ratios (you do know it is molar, not ng ratio!) of insert:vector will mean a lot less insert than you are using.

For pJET, the recommended  PCR product quantity to obtain 0.15 pmol of DNA ends is 25-50 ng for products of 500-1000 (my product is 700 pb). Is not mentioned if is purified PCR product or not.

bob1, on 01 February 2013 - 02:53 PM, said:

With 10 ng of control transformation you should be getting several hundred colonies on the plate, there may be a problem with the transforamation efficiency of your bugs.

In my transformation control I get 200+ colonies (I use 1 ul of  miniprep purified pGEM with an insert); quantity I don't get using my ligation product. Just few hundreds would be enough.

### In Topic: Problem with ligation using pJET and pGEM for clone library

01 February 2013 - 11:26 AM

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How much DNA are you ligating?

Well I have been using 16-18 ng of purified PCR product during ligation. I'm getting low yield during purification.

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What ratios of insert to vector have you tried?

I always used 3:1 Insert:vector (pJET and pGEM)

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Is the ligation buffer fresh (contains ATP which degrades rapidly)?

The buffer used for pJET ligation is fresh. The one for pGEM, not so fresh.

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How much control DNA are you transforming (in ng)?

19,16 ng of purified pGEM

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How much of your ligation are you using for the transformation (and how big a volume of cells)?

I have tried using 5 ul and 10 ul of the ligation product in 100 ul of chemocompetent DH5-alpha