How do I determine the optimal gene-to-vector ratio for ligation?

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In summary, the conversation discusses the process of ligating a gene fragment into a Pbluescript plasmid. The fragment and vector have both been digested and purified, but the concentration ratio of the gene to the vector is crucial. The recommended ratio can vary from 8:1 to 1:8 and it is important to find the best ratio through experimentation. The conversation also mentions a helpful website for seeking advice on molecular biology protocols.
  • #1
rockind78
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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
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
Thank you Ian. I appreciate the quick response. I will look at my protocols again to see if I missed something.
 
  • #4
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
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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1. What is the purpose of ligating a gene fragment?

Ligating a gene fragment involves joining two pieces of DNA together using an enzyme called a ligase. This is often done in genetic engineering to create recombinant DNA or to insert a specific gene into a plasmid for cloning purposes.

2. How is a gene fragment ligated?

A gene fragment is typically ligated by first cutting it with a restriction enzyme to create sticky ends. The plasmid or DNA vector is also cut with the same restriction enzyme to create complementary sticky ends. The two pieces are then mixed together with ligase, which joins the sticky ends together to form a complete DNA molecule.

3. What factors can affect the efficiency of gene fragment ligation?

Several factors can affect the efficiency of gene fragment ligation, including the concentration and activity of the ligase enzyme, the length and sequence of the sticky ends, and the purity and quality of the DNA fragments being ligated.

4. How can I confirm successful gene fragment ligation?

Successful gene fragment ligation can be confirmed through various methods, such as gel electrophoresis, DNA sequencing, and restriction enzyme digestion. Gel electrophoresis involves separating the DNA fragments by size, while DNA sequencing can confirm the sequence of the ligated gene. Restriction enzyme digestion can also be used to confirm the presence of the gene fragment in the ligation product.

5. Are there any precautions I should take when ligating a gene fragment?

When ligating a gene fragment, it is important to work in a sterile environment to prevent contamination. It is also essential to use high-quality, purified DNA fragments to ensure successful ligation. Additionally, following the recommended protocols and using the appropriate amounts of each component can help increase the efficiency of gene fragment ligation.

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