My DNA insert size is 746bp. I have designed the primers with the EcoR1 and Sal1 enzyme sites at 5'.
look for induction with IPTG (time) and Bl21DE3 Lys cells for your pGET4T1 vector. as for steps follow standard procedure for linearization and ligation of vector and insert in proper ratio (Insert: vector) depending on your insert size. follow the ligation procedure further and select colonies desired upon selection based on vector information.
The basic steps would generally be as follows:
1) Make sure you have designed your primers such that the 5' end of your construct will be in frame with the upstream GST and thrombin site. (And inlcude a stop codon in your reverse primer.)
2) Set up a digest of the vector with the respective restriction enzymes, in your case EcoRI and SalI.
3) PCR amplify your insert.
4) Purify both your vector and insert on agarose gels and extract from the gel bands (generally done with commercial kits these days). I would suggest using a 0.7% gel for the vector due to its size.
5) Digest the insert with the same enzymes and use a kit (e.g. a PCR purification kit) to remove the enzymes and the small stubs removed from the insert by digestion.
6) Determine the concentrations of the vector and insert.
7) Set up a ligation - there are very good instant/quick ligases available from e.g. NEB - using a 3:1 molar ratio of insert:vector.
8) Transform into competent cells of a cloning strain of E. coli you have available and plate on selective plates.
9) Miniprep some clones and verify by sequencing.
Before the ligation reaction I checked my vector backbone and the insert size on 1% agarose. But I got very faint bands for both the vector and the insert. Any suggestion how much of insert : vector I should be taking in case both are showing the faint bands in the gel. It would be helpful for me if I have the ligation recipe for faint bands (insert and vector)
If you have very faint bands, chances are low that the ligation reaction will give you enough product for a successful transformation. For subcloning we usually use about 2 microg of the plasmid which is digested, dephosphorylated, gel purified and eluted with a minimal amount of elution buffer, from this eluate we use between 0.5 and 1 microl for a 10 microl ligation reaction.
For the insert use an amount which is approximately 2 microg of insert for the digest, i.e. you have to use more when the DNA is in a plasmid, gel purify and elute with a minimal amount.
I don't check the DNA concentrations for eluates because the concentrations are too low to be accurately read. Playing with the plasmid:insert ratio never changed anything for me, either it works (95% of the time) or it doesn't, for a 10 microl ligation I reaction I just add the insert to the give the final volume (no additional water needed).
If you are having problems with getting enough vector, I would simply go back and perform the digestion again with more input material. If you digest 5-10 µg of vector, this should be plenty to see an intense band on an agarose gel - in fact I would run it in 2-3 adjacent lanes and pool them. If you then extract the band with a kit and elute into e.g. 30 µl, you should easily be achieving 25-100 ng/µl. If you use 1-2 µl of this in a reaction it will be plenty.
For the insert, if you are having a low yield on a gel, then I would go back and try to optimise your PCR reaction. This could involve redesigning the primers, optimising the annealing temperature (e.g. using a temp gradient step) and the extension time. You could also up the number of cycles to 35 with a good proof-reading polymerase. Again, gel purify and elute in a small volume to get a high concentration.
I always check the concentrations of the vector and insert using a Nanodrop machine - this has a small path length so can accurately determine quite low concentrations. I have always found that you will have a much greater chance of success if you start with higher concentrations of vector and insert.
For 50 ng of vector you will want 50 * 3 * (745 / 4969) = 22 ng of insert. This should be easily achievable, often corresponding to 1-2 µl.
If you are using a standard T4 ligase I would perhaps try using a high concentration stock. You can also try RT for an hour or 16C overnight. However, I would recommend one of the quick/instant ligation mixes available as they make life easier, save a day compared to overnight ligation, and tend to "just work".
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