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concentration for cloning


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#1 sahil

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Posted 12 January 2010 - 10:08 AM

Dear all ,
I am trying to clone a 1400 bp sequence in RISAP vector digested by ClaI , insert is also digested by ClaI . Now I have to make them to ligate , but the concentration of insert is 3.3 ng/microl and vector is 6 ng/microl . I want to ask whether this concentration would be sufficient for cloning ?
please let me know , i would highly appreciate your help

Regards
Sahil

#2 pDNA

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Posted 12 January 2010 - 11:12 AM

normally u use up to 100ng of vector and calculate the amount of insert you need to have eg. for a 5 molar excess of the insert. The calculation is done according to this formula:

insert mass in ng = 5 X (insert lenght in bp/vector lenght in bp) X vector mass in ng

Normally a ligation is done in a 20 or 10Ál scale (most of the time the smaller the better!) ...i think that your concentrations will farly exceed this volume due to the low concentrations.

Additionally, if the readings of the photometer are that low you are in danger that you are measuring background noise! ...so i would recommend to start from the scratch or if you have a large volume of your solution (around 50Ál) and you verified your concentrations than you could try precipitate your DNA and resuspend in a small volume to increase the concentration.

Regards,
p




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