5 replies to this topic

[beulah]

on what basis does a pair of restriction sites be choosen for directional cloning???

[T C]

Hey,

You have to be more specific as to what do you want to do.

I generally prefer to engineer a Nde I or Nco I at the start since they contain the ATG codon.  At the end, it depends upon the frame.  Like for pET series of vectors, Hind III is in frame with the Cter His tag, Eco RI is -1 and so on.  This is when you want to express the protein; for general cloning, it hardly  makes any difference and you use some good cutters and make yr strategy that way.

Hope it helps.

best,
TC  

beulah, on Jul 9 2009, 07:35 PM, said:

on what basis does a pair of restriction sites be choosen for directional cloning???

[fishdoc]

beulah, on Jul 9 2009, 08:05 AM, said:

on what basis does a pair of restriction sites be choosen for directional cloning???

A few things to keep in mind:

1) make sure the digested ends aren't compatible with each other
2) choose enzymes that have 100%  efficiency in the same buffer (if possible, at least 75% for sure)
3) make sure the enzymes are compatible for double digestion (this goes along with 2)
4) make sure your restriction sites are not within the DNA you intend to amplify

There are probably a few more helpful hints out there, but these are the ones I go through immediately when planning directional cloning.

[Curtis]

can anyone give a link to any website with full details on how to design the restriction sites or the overhang nucleotides before them?

I usually use Fermentas or NEB's tables to determine how many nucleotide overhang I must have for certain enzymes.

sometimes I have only 1-2 extra nucleotide overhang before my RE site, but my supervisor says to go for 6-8 extra overhangs. maybe that's why my cloning is not always efficient !?

[Rsm]

Curtis, on Jul 11 2009, 08:04 AM, said:

can anyone give a link to any website with full details on how to design the restriction sites or the overhang nucleotides before them?

I usually use Fermentas or NEB's tables to determine how many nucleotide overhang I must have for certain enzymes.

sometimes I have only 1-2 extra nucleotide overhang before my RE site, but my supervisor says to go for 6-8 extra overhangs. maybe that's why my cloning is not always efficient !?

You mentioned the NEB table here, so I guess you know what you're talking about. I usually use TTTT in front, then RE site (6nt) and then 20nt of ORF. That makes 30nt total, so I can order the cheapest/smallest amount of primer ($7.80 per 30mer, I guess that's a competitive price). So far it works for any RE (well, Kpn1/EcoR1/BamH1/Hind3/Nhe1/Nco1/Apa1/Pst1/Xho1/Not1 at least).
Cheers,
Minna

[fishdoc]

Curtis, on Jul 11 2009, 10:04 AM, said:

can anyone give a link to any website with full details on how to design the restriction sites or the overhang nucleotides before them?

I usually use Fermentas or NEB's tables to determine how many nucleotide overhang I must have for certain enzymes.

sometimes I have only 1-2 extra nucleotide overhang before my RE site, but my supervisor says to go for 6-8 extra overhangs. maybe that's why my cloning is not always efficient !?

I usually use 6 nts outside the RE site. Have never had a problem with that number. A couple years ago, the NEB catalog recommended that for a number of enzymes, but the latest volumes have dropped it down to 1 or 2 for most. For determining which are good for double digest, I use this site: http://www.neb.com/n...tCalculator.asp