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cloning - (Apr/10/2010 )

hi friends,
I am trying to clone my inserts of size range 300bp-500bp in pZErO vector. I am getting very few colonies. Among those few contain the desired inserts while rest are empty.The property of vector is such that only recombinants grow. I have tried varying insert:vector ratios but facing same problem. The efficiency of transformation and vector has also been checked and found to be good. i have used phenol choloform during precipitation of my inserts. Does this has any effect in cloning?

-mlu3-

What restriction enzymes do you use? ...have you checked if they work properly?

In such cases it is good to do an extra ligation and put the whole thing on agarose gel to check if you get ligation products. If you dont see ligation products something is wrong with your enzymes!

What are your spectrophotometric readings of your insert DNA treated by chloroform/phenol? Do you hava a high 260/230? Maybe residual phenol is your problem (i do not use this to purify my inserts ...we do spin colum purification ...but i can imagine easily that phenol is a problem for restriction enzymes or ligases).

Regards,
p

-pDNA-

pDNA on Apr 10 2010, 01:43 AM said:

What restriction enzymes do you use? ...have you checked if they work properly?

In such cases it is good to do an extra ligation and put the whole thing on agarose gel to check if you get ligation products. If you dont see ligation products something is wrong with your enzymes!

What are your spectrophotometric readings of your insert DNA treated by chloroform/phenol? Do you hava a high 260/230? Maybe residual phenol is your problem (i do not use this to purify my inserts ...we do spin colum purification ...but i can imagine easily that phenol is a problem for restriction enzymes or ligases).

Regards,
p



ok 160/180 is 1.78 and 260/230 is 0.17. Test inserts have been provided to check the function of vector and when i did their ligation i got good number of colonies with the desired inserts. I am just not getting the results with my concatemers. i will try to remove the possibility of phenol being the culprit. Thank you so much for your suggecstions.
bye

-mlu3-

Your 260/230 ratio indicates that there is residual phenol ...it should be above 2 for pure samples!

Additional Phenol contamination can wreck your estimation of DNA concentration since it also absorbs at 260 nm ...have you checked your insert concentrations on an agarose gel?

If you do blunt end ligation impurities can be a major cause for failed ligations ...i would recommend repurification.

Regards,
p

-pDNA-

mlu3 on Apr 9 2010, 11:27 PM said:

hi friends,
I am trying to clone my inserts of size range 300bp-500bp in pZErO vector. I am getting very few colonies. Among those few contain the desired inserts while rest are empty.The property of vector is such that only recombinants grow. I have tried varying insert:vector ratios but facing same problem. The efficiency of transformation and vector has also been checked and found to be good. i have used phenol choloform during precipitation of my inserts. Does this has any effect in cloning?


Blunt end cloning is never fun, but once you get a good estimation of getting good quantification of your insert it works really well. You need to be as close to the real amount of DNA as possible, running out on 0.8% agarose gel works really well, with a molecular weight marker.
Don't forget that your molar ratio not only affects insert ligating to vector but also more than one insert may get ligated and inserted into vector.
Instead used a T-vector (e.g. pGEM-T), use a regular Taq polymerase that will leave out A-overhangs. This works much better even if your molar ratios are not accurate.

-DRP-

DRP on Apr 11 2010, 07:49 PM said:

mlu3 on Apr 9 2010, 11:27 PM said:

hi friends,
I am trying to clone my inserts of size range 300bp-500bp in pZErO vector. I am getting very few colonies. Among those few contain the desired inserts while rest are empty.The property of vector is such that only recombinants grow. I have tried varying insert:vector ratios but facing same problem. The efficiency of transformation and vector has also been checked and found to be good. i have used phenol choloform during precipitation of my inserts. Does this has any effect in cloning?


Blunt end cloning is never fun, but once you get a good estimation of getting good quantification of your insert it works really well. You need to be as close to the real amount of DNA as possible, running out on 0.8% agarose gel works really well, with a molecular weight marker.
Don't forget that your molar ratio not only affects insert ligating to vector but also more than one insert may get ligated and inserted into vector.
Instead used a T-vector (e.g. pGEM-T), use a regular Taq polymerase that will leave out A-overhangs. This works much better even if your molar ratios are not accurate.


hi, i would definitely consider your suggestions. I am doing sticky end ligation, my inserts and vector have 4 bp overhangs namely""CATG". I require maximum efficiency of cloning. I won't be able to check my inserts on gel because that amount would go waste. i tried molar ratios like2:1,3:1,4:1 but my problem is i am getting very less number of colonies. thank you

-mlu3-

hi, i would definitely consider your suggestions. I am doing sticky end ligation, my inserts and vector have 4 bp overhangs namely""CATG". I require maximum efficiency of cloning. I won't be able to check my inserts on gel because that amount would go waste. i tried molar ratios like2:1,3:1,4:1 but my problem is i am getting very less number of colonies. thank you



Ligation reaction only requires picograms of DNA, so unless you have less than 1ng of insert you should be able to run out 1ul on gel to get an estimate of quantity. Another option is to use nanodrop, if you have access to it. I don't really trust regular spectrophotometers with such low amount of DNA quantification.
On another note, getting less colonies may have nothing to do with your molar ratios, it may be a case of adding too much DNA to competent cells that inhibits the transformation efficiency. Are you using home made competent cells or commercial ones ? they specially tell you how much DNA to use/transform...too much is not always good.

-DRP-