21 replies to this topic

[slapolla]

Thanks so much badcell! When I have a construct that I can use that trick for I will try it and let you know how it goes.

[Dr.G]

Hi,
I agree that the concept is clever.

However, all considering, isn't it just as easy to dephosphorylate - just in case? Using SAP, you can add 5 Units (let's say) the last 15 min of your RE incubation, then heat inactivate. This should take care of all your single cut plasmids.

[badcell]

I'm not a big fan of dephosphorylation, as SAP has munched away my DNA several times, and I know from other people that this can happen quite often. Also, dephosphorylation, when it works, is not 100% efficient. Just as after overnight incubation with an RE not all your plasmid molecules would have been cut, after the much shorter 15 min incubation with SAP/CIP/wathever, not all your digested molecules would be dephosphorylated. After saying that, I must say that I usually dephosphorylate my vector DNA extensively, for 30 min-1h, but I always keep an aliquot untreated... just in case SAP is hungry.
Cheers

[xiaoxiaoPROXY8891]

wow.. im in highschool and i have no idea what anyone is talking about. haha. it sounds outrageously intresting though

[lucilius]

In theory it's not absolutely necessary to dephosphorylate if you are using two REs with cohesive ends, but in fact, if the restriction efficiency of the enzymes is not 100%, you will get some molecules which have been cut only with one or the other enzyme, so they will religate if they are phosphorylated. One useful trick when cloning using two different sites is checking if you loose any site in the vector multiple cloning site (if any site is between the two REs you are using and not anywhere else in the vector). If this is the case, and this site is not found neither in your insert, you can digest your ligation with that enzyme and you will cut only your religated plasmids and not the ones with your insert, which will not have that site. So you will get only colonies with the insert.

Could someone explain this?

If I understand it right badcell means that you will not cut the vector with an insert because the insert is "between" the restriction site of that enzyme or?

Eg. TT/GG is where the enzyme cuts and you have inserted something there, so you have TTAA..AAGG so that the enzyme cant cut there anymore?

Or?

Another question: if you cut a vector with 2 RE, then wont you destroy the vector? Or are both RE cutting at +- the same place so that if one cuts, the other one cant cut there anymore?

[chaperone123]

Hi Badcell,
well good news finally. i got 0 colonies in my no-insert control ligation plate. and around 30-40 colonies in my other transformant plates. ur suggestion worked real goood!! i like to mention tht i did not heat inactivate the ligate mixture. i added 0.5ul of rest enz+10ul enz buffer. added 1ul to the ligation mixture and incubated for 15mins at 37C. then transformed.
its better tht u too try without deactivation liagase as u may also get 30 colonies instead of 3!!! good for u. u saved my day.
cheeeeeeeers.
rajgene

Hello Rajgene,

Can you please explain little in detail regarding the digestion of ligation mixture you did. If I had understood properly you will setup ligation overnight at 16 C and take 1 microleter from this ligation mixture and add 1 microleter of RE and 10 microleter of 10X RE buffer? SO total volume will be 11.5 microleter. Don't you think the buffer concentration is bit high for this digestion. Because generally you use 1 microleter of 10X buffer to total volume of 10 microleter.
And how much volume from this mixture you take for transformation?

Thanks in advance

[Bpaul]

Hey Badcell!!

I am an absolute beginner in molecular cloning! I have been going thru this thread and came across your idea to digest the ligation mixture!It sounds great and am about to try it this evening!



Thank you!!!

Edited by Bpaul, 29 July 2012 - 09:22 PM.