Dear all, , I want to know what the best basic theory we need use rasio 1:90 between vector : dna with using p.BluecriptSK-II digested by EcoRV?
best regards,
Dewi Yuliana
Dear Yewi,
I'm not quite shure that I understood your question correctly but if you need a reasoning for vector to insert ratio, it highly depends on your insertlength. To calculate the optimal ratio I always used this formula provided with the pGEM-T easy Kit:
((ng of vector*kb size of insert)/kb size of vector)*insert:vector molar ratio = ng of insert
Hope it helps
Best regards
Dear Yewi,
I'm not quite shure that I understood your question correctly but if you need a reasoning for vector to insert ratio, it highly depends on your insertlength. To calculate the optimal ratio I always used this formula provided with the pGEM-T easy Kit:
((ng of vector*kb size of insert)/kb size of vector)*insert:vector molar ratio = ng of insert
Hope it helps
Best regards
For the ideal ligation situation it is better to have a same molar ratio i.e. same number of molecules for both vector and insert. If one of the molecules (vector) is much bigger than the other, the total mass of it's DNA will be bigger as well.
For example you want to have in your ligation 1 nmol of vector and 1 nmol of insert, which is 6x10^14 molecules each. Say the mass of 1 molecule of insert DNA is 1 ag, and 1 molecule of vector is 90 ag. So the mass of 1 nmol of insert will be 1x6x10^14 ag, and for vector 90x6x10^14 ag.
Also people often take up to 3 fold or so excess of the insert.
Do not forget to dephosphorylate your vector.
EcoRV gives blunt ends and ligation will be poorly efficient (perhaps 1% compaired to cohesive ends). It is recommanded to use a molar ratio of 2 to 10 (calculated according to insert size as explained above) because the phosphatase that will prevent self-ligation will not be 100% efficient (we like the Rapid alkaline phosphatase because it does not damage the DNA). So using less insert will produce many colonies with empty vector, while using too much may reduce the number of colonies and give colonies with more than one insert.
Good luck,
Dear All,
thanks very much for your reply, could you mind share with me your experience use blunt end ligation? what kind molar rasio did you use? and what kind approach do you to over come the low efficiency ?
Among things that can improve it are: longer incubation times, smaller ligation volume, adding PEG up to 10% and briefly centrifuging before the incubation. Efficiency may still be low, so you will need to screen many colonies. But in your case it is easy, just use plates with X-gal.
My personal strategy: if blunt end ligation does not work at the first attempt, while controls are OK, try another strategy, as they are always alternatives with cohesive ends, PCR, etc... There are also possibilities without ligase. A good reading: Matsumura BioTechniques 59:IV-XIII (September 2015) doi 10.2144/000114324
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