Single Restriction Site cloning - Cloning (Oct/04/2010 )

Hello,

I have the insert as a plasmid construct between EcoRI and Not I restriction sites. I need to clone it into another vector with only EcoRI site and no other site. Could someone please advise as to what methodology needs to be used. I have always used MCS never a single site so unsure as to what should be the steps. Any help and references would be appreciated.


thanks

Shini

Shiny on Mon Oct 4 21:16:06 2010 said:

PCR out the gene using Forward Primer (ECOR1 + sequence homology) and the (Reverse Homology + ECOR1 Site).

Use a program like Primer3 to get your homolgy then just add the EcoRI sites onto it. Google "Primer3"

Now, if you add some extra crap on both ends you can digest the PCR product directly. Or you can just TA clone if using TAQ. I prefer adding about 4 bases onto each end so I can digest the PCR product directly.

you should first PCR up the gene using primers for either end of the gene, with EcoRI sites added, so the first 20 bases of your gene, then an EcoRI site in front of that, than some random 4-6 bases in front of that to make sure the RE will cut properly. Then do the same for the end of the gene, remembering to do reverse complement, so:

CATG GAATTC FIRST-20-BASES-OF-GENE

LAST-20-BASES-OF-GENE CTTAAG CATG


Then purify that PCR product

Cut both the PCRed up gene and the vector with EcoRI, in seperate tubes obviously. However you must get hold of some Alkaline Phosphatase to use on the cut vector, this removes the phosphates from the ends of the cut DNA and prevents the vector re-ligating back to itself. This isn't neccecery when doing digest with 2 different enzymes, as the different sites cannot re-ligate to each other, but if you don't do this step when only using 1 enzyme you will get lots and lots of empty-vector colonies as the EcoRI sites re-ligate.

Don't use the Phosphotase on the insert however, otherwise you will not be able to get anything! When the insert ligates to the vector, the phosphates on the insert can be used, if you phosphorylate them then you wont ligate anything!

Then just do the normal gel-purification, ligation, transformation, etc.


I did a very similar thing to what you are doing now a few weeks ago, I have a couple of topics I posted on the topic in this forum from 2-3 weeks ago if you want some help!

I also found this guide very useful:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cloning_guide.asp

Thnks philman n Jordan for the advice.. I do not think it wud b an option 2 pcr d insert to introduce the xtra ecorI site n subsequent Pcr as its quiet a large insert.. to b apt, its a gene already cloned into a vector, wich needs to b subcloned into a new vector for xpression wich is nt open fr manipulation. What do u guys thnk abt blunt ended ligation??

Also philman could you please provide the topic of d previous thread u talking about.

I don't think blunt ended ligation would be neccecery, you could cut the insert out using your two enzymes, EcoRI and NotI, then create a short pair of oligos which annealed together to form a NotI site at one end and an EcoRI site at the other. So assuming the NotI site is at the 5' end of the gene:

5' GGCCGC catg G 3'

3' CG gtac CTTAA 5'

then cut the insert out with NotI and EcoRI, purify it, then ligate that annealed linker onto the insert. It will ligate to both ends, but then cut with EcoRI again and you will be left with your gene with EcoRI sites at either end.

I don

@Philman it does sound like a plan.. but then I see two problems here with this approach

1.ligating a large chunk of DNA (say few Kbs) and a few nucleotides longer linker.. What ratio's do you normally take for your ligation? Is there like a formula or something that you could use for setting up ligation reaction?
2. How would you screen the positive clones, reason being the two new EcoRI sites would be quite closely placed, so definitely restriction analysis would not be an option.


thanks for the response