9 replies to this topic

[Procyon]

Hello,

I'm trying to clone a PCR product of 750 bp amplified from DNA extracted from environmental samples. I used a "Wizard PCR and Gel clean-up system" to purify the gel band of my product (I'm always careful to expose the gel to UV less time as possible). For transformation i'm using chemocompetent DH5-alpha, with a heat-shock of 42°C at 45 seconds.

I'm using pGEM or pJET as vector, but I get less than 40 transformant colonies (but I need 100+ for clone library). In my transformation control I get always 100+ colonies ( I use 1 ul of  miniprep purified pGEM with an insert) ; but when I use a ligation control for pJET, I also get 40 or so transformants.

I've previously done the ligation and transformation using pJET and DH5a with a fragment of 370 bp also amplified from environmental DNA; obtaining 647 transformants with my insert.

I suspect there is a problem with ligation, based on the controls. Is there an upper limit of insert DNA (purified or direct from PCR) quantity during the ligation? Should I increase the time of ligation?

I've repeated the PCR + ligation + transformation changing different parameters, but I still get less than 40 (white when i use pGem) colonies. I really need some advice of what to do or what may I have forgotten.

Edited by Procyon, 31 January 2013 - 07:59 AM.

[bob1]

How much DNA are you ligating?
What ratios of insert to vector have you tried?
Is the ligation buffer fresh (contains ATP which degrades rapidly)?
How much control DNA are you transforming (in ng)?
How much of your ligation are you using for the transformation (and how big a volume of cells)?

[Procyon]

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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.

Quote

What ratios of insert to vector have you tried?

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

Quote

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.

Quote

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

Edited by Procyon, 01 February 2013 - 01:39 PM.

[bob1]

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 nsert than you are using.

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.

Typically you want to keep the amount of ligation mix below 5% of the volume of bacteria you are transforming as the DTT in the ligation mix inhibits the bacteria somehow (at least this is how I understand it), so 5 ul in 100 should be fine.

[Procyon]

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.

[phage434]

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.

[Procyon]

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.

Quote

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'm a bit confused about this. These molar ratios are of the final ligation mix?

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?

[phage434]

If you have 50 ng of a 100 bp fragment, that this 50x as many fragments than if you have 50 ng of a 5000 bp fragment. Ligation is best when there is approximately the same number of vector and insert fragments, so you have to scale the amount of DNA according to fragment length; that is, you need about equimolar amounts of each fragment.

[bob1]

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.

[Procyon]

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.