Ligate a gene fragment

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I will be attempting to ligate a gene fragment (~650bp's) into a Pbluescript plasmid here in the next day or so. The fragment and the vector have both already been digested and purified, so that's not an issue. However, I have been advised by a number of differnt people that the concentrations of my gene relative to my vector are VERY important and I do not know what this relationship is supposed to be. I looked in the Short Protocols Molecular Biology and did not find anything. Does anyone have any good links or words of wisdom? Thanks!

Dustan
 

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  • #2
iansmith
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There a ratio that you should follow according to the protocols. Ratio can vary from 8:1 to 1:8 (plasmid:insert).

Here the formula to calculate the amount needed

ngofvector × kbsizeofinsert × insert:vector molar ratio = ng of insert
----------------------------
kb size of vector

You usually have to work you ratio out. What we do in the lab, we set 3 or 4 differrent reaction, we keep the plasmid volume the same (usually 1 uL) and we use a range of volume for the insert (usually 1 uL, 2uL, 4uL). Then we see what works best and reuse it if necessary.
 
  • #3
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Thank you Ian. I appreciate the quick response. I will look at my protocols again to see if I missed something.
 
  • #4
Monique
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I think the usual thing is 1 vector : 3 insert.. but the best thing is to try several at the same time and see which one gives you the best enrichement (as Ian said).

Try one ligation without insert (to see self closing vectors) and then you could do 1:3, 1:5, maybe 1:7 and 1:9
 
  • #5
Another God
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As for a link: http://micro.nwfsc.noaa.gov/forums/index.php?sid=5a9ee6c992acccf932b83f1b3aa98383 is the only website that I have ever found that seems to be dedicated to helping people figure out questions like that (And i think I only found this website becuase Ian pointed me towards it many many months ago).

It's a nice website in theory, Forums like these where people ask questions and get answers, unfortunately it isn't as active as you would like, and so you aren't guaranteed a response.
 
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