I had done PCR amplification of my gene of interest using bacterial genomic dna as template. Further gel extraction (thermo scientific) of 200ul pcr reaction volume had given pcr product having concentration 24.78 ng/ul (total volume 30ul)- Measured through nanodrop. Its my first time to proceed with small concentration of pcr product. What you would suggest a further Gel extraction from large volume of PCR reaction or I will move on further? I think after restriction digestion performing further gel extraction will reduce the concentration of Insert. So is it feasible to move for restriction digestion or I will Setup a large volume PCR reaction to get more concentration PCR product?
Hi Vishal,
You could skip the second gel extraction after the restriction digest step and try to purify the insert with a precipitation step instead. You might recover more insert this way than gel purification. However, if you are more comfortable with your current method I would suggest repeating your PCR and combining multiple gel extractions prior to the digest step and 2nd gel purification step. Another suggestion depending on your resource is trying silica column purification following the PCR and RE step. You may loose less of your PCR products this way.
You have about 750ng of insert here. You will likely lose 50% in the second gel clean.
I generally use 100ng vector in a ligation reaction so depending on what size your vector and insert are and what ratios you want to try for ligation, you can work out whether you will have enough.
Vishal, you will need to estimate concentration of your PCR in pM (or any other mollarity) since ng/ul does not tell you how many molecules you have. You will have to estimate the ratio between your vector and insert (usually 3:1. Then you have to set up the following reactions - 1. Vector (no ligase), 2. Vector + Ligase, 3.Vector + Ligase + Kinase, and finally 4. your Insert+ Vector+ Ligase. Transform 1/10 of the ligation mix of each experiment , plate and in 24h count the colonies in each plate.
Hope that helps
i also think that you should not go for second gel extraction as you have 750 ng of insert . You should go for digestion and directly purify it by using the same kit but finally elute it in less volume and then i think you will have sufficient amount of insert DNA to use for ligation. This is based on my experience as i did the same few days ago...gud luck
I agree with the plan of concentrating the insert DNA after your digestion. If you still don't have enough insert to proceed, I would suggest setting up several smaller volume PCR reactions (perhaps 10 reactions of 20 ul each). I found that our PCR machine will give a better yield from several small reactions than from 1 large volume reaction.
If you think the PCR yield is not good enough, try optimizing the PCR reaction(buffer-co factor concentration for example). I would also agree as others, since you need very little amount of your digested insert (not even 50ng required!). Good luck.
Ialso agree with this point that our PCR machines give good yield with smaller volumes (10 or 20ul) so you can also try this if you want more insert...otherwise u should go ahead with this amount also as i told earlier ....
I performed the restriction digestion on obtained insert and further purified it from gel and gel extracted in 20ul reaction volume. When i run it 5 ul of each- insert as well as double digested vector their is only insert band was visible, however i performed the reaction for ligation measuring concetration in 15ul reaction. But after transformation no colonies are visible. suggestions needed.
How far are the restriction sites from the 5' and 3' ends? Instead of gel extraction you could use a PCR purification column to purify the digested insert if the cleaved ends are
You could repeat the PCR and do the restriction digest prior to gel purifying, then you only have to purify once.
Though it will depend upon the size of insert, I think the amounts which you have are more than sufficient for any cloning. Even if you loose 50%, there will be enough PCR amplicon for putting in to a vector and getting a clone. Just keep a track of the PCR DNA concentration at each step, and the calculations in order to use a proper ratio of vector vs PCR amplicon.
Vishal, without proper controls (that I described in the earlier post) is very difficult/impossible to interpret your results.
Petere Sir.. I will perform experiments once again with control as you suggested and will inform you soon. Thanks to everyone for your suggestions.
Ciaran Lee Sir... As i think you are asking the exact position of both restriction sites from 5' and 3'/ My gene is 1.5 KB long and PCR purified product contains 9bp overhang, restriction site and then gene same as 3' gene contains termination codon then restriction site (6bp) then 9bp overhang.
you are saying that when you run gel your insert was visible but your double digested vector wasn't am i right ???............if this is the case then i think you should work on your vector double digestion......can you plz explain me clearly so that i can help you.....
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