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Ligation/Transformation problems & 260/230 ratio of vector


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#1 geraldgsw

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Posted 15 September 2009 - 03:40 AM

Hi everyone, I'm having trouble with my ligation/transformation and I think maybe it has something to do with my vector. After digesting and dephosphorylating it and doing clean-up using Qiagen Gel Extraction kit (using 3 volumes of buffer QG, 1 volume of isopropanol and starting from step 6), my yield is around 50 ng/ul, 260/280 ratios are around 1.8-1.9 but 260/230 ratios are around 0.3. I repeated my digest/dephosphorylation/cleanup but I got similar values for 260/230 ratios. Can this cause ligation or transformation to fail? Is there anything I can do to increase the purity of my vectors?

After doing ligation and transformation, I keep getting smeary streaks of bacteria. I've changed the plates and made new Ampicillin from powder, and my next step is to use a new batch of competent cells because my negative control plate had smears on it also (my colleagues say perhaps this is because I plate too many cells, can this be true?) But I thought I should ask about my vectors as well. Also, is there anyway to check if the problem lies with ligation or with transformation when my insert is 90bp but my vector is 12kbp?

Many thanks in advance!

#2 fishdoc

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Posted 15 September 2009 - 05:21 AM

Hi everyone, I'm having trouble with my ligation/transformation and I think maybe it has something to do with my vector. After digesting and dephosphorylating it and doing clean-up using Qiagen Gel Extraction kit (using 3 volumes of buffer QG, 1 volume of isopropanol and starting from step 6), my yield is around 50 ng/ul, 260/280 ratios are around 1.8-1.9 but 260/230 ratios are around 0.3. I repeated my digest/dephosphorylation/cleanup but I got similar values for 260/230 ratios. Can this cause ligation or transformation to fail? Is there anything I can do to increase the purity of my vectors?

After doing ligation and transformation, I keep getting smeary streaks of bacteria. I've changed the plates and made new Ampicillin from powder, and my next step is to use a new batch of competent cells because my negative control plate had smears on it also (my colleagues say perhaps this is because I plate too many cells, can this be true?) But I thought I should ask about my vectors as well. Also, is there anyway to check if the problem lies with ligation or with transformation when my insert is 90bp but my vector is 12kbp?

Many thanks in advance!




I rarely get higher than 0.5 for 260/230 following gel purifications with the Qiagen kit, get similar ng/ul values, and have no problems with ligations. I'm not sure what it is in the cleanup, but the 260/230 is ALWAYS poor for me, sometimes as low as 0.03.

My guess is the competent cells, seeing as the controls have the same problem. And plating too many cells can be a problem, too. You can overwhelm the antibiotic and everything will grow. In fact, now I think I know what you mean by smeary streaks - there are patches of bacterial lawn on the plate. Definitely from spreading too much.

My guess is there is no need to check the ligation, lower the amount of cells first.

#3 Michaelro

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Posted 16 September 2009 - 01:11 AM

Hi
260/230 ratio is the indication for amount of salts in the eluted DNA.
Higher ratio means higher purity.
Usually it is not a problem for ligations.
Furthermore, extra wash may help.
Your problem seems to be the transformation.
However, I need to see your transformation protocol in order to help
Best
Michael

#4 geraldgsw

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Posted 16 September 2009 - 01:32 AM

thanks fishdoc and Michael for your replies.

Michael: My transformation protocol is as follows.

Add 2.5 ul to 10 ul of cells. Incubate on ice, 30 min. Heat shock 42 degC, 45 seconds. Return to ice, 2 min.
Add 0.9ml SOC, shake 37 degC at 300rpm for 1 hour.
Spread on LB-Amp100 plates.

Thanks!

#5 Michaelro

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Posted 16 September 2009 - 07:11 AM

thanks fishdoc and Michael for your replies.

Michael: My transformation protocol is as follows.

Add 2.5 ul to 10 ul of cells. Incubate on ice, 30 min. Heat shock 42 degC, 45 seconds. Return to ice, 2 min.
Add 0.9ml SOC, shake 37 degC at 300rpm for 1 hour.
Spread on LB-Amp100 plates.

Thanks!


Hi
Try to spread 100ul of the cells.
Spin the rest on 1000G for 5min.
Remove the sup - leave only 100ul.
Resuspend the pellet in 100ul and spread on another plate.
Maybe it will solve the smear problem.
Good Luck




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