21 replies to this topic

[rajgene]

Hi all,
i am digesting my vector with 2 diffrent rest enzymes(Bgl II + Mlu I) yeilding diff sticky ends. after digesting do i have to proceed to dephosphorylation or can i skip it. in theory the sticky ends generated above would prevent self-ligation right?
i want to know is dephosphorylation is absloutely essential in such cases.
thx
rajgene

[badcell]

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.

[rajgene]

badcell, on Jan 26 2005, 11:41 AM, said:

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.

Wow....wht a trick. badcell is quite good..

[badcell]

Yeah, that dirty little trick has spared me a lot of minipreps...

[zhgljj1998]

badcell, on Jan 27 2005, 03:52 AM, said:

Yeah, that dirty little trick has spared me a lot of minipreps...

Digest the Ligation reaction. man, you are good. by the way, about the Miniprep, actually, how much DNA can you get from one miniprep? sometimes, i got very low concentration. is there any trick? thanks.

[badcell]

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how much DNA can you get from one miniprep? sometimes, i got very low concentration

I usually get around 20-40 micrograms DNA from a 3-5 ml overnight culture, using qiagen columns or something of the sort. The quantity of DNA will vary depending if your plasmid is low-copy or high-copy. One good thing is not to overload the column, use 3-5 ml for a high-copy plasmid, or 10 ml for a low-copy. This is a case of less is more. Cheers!

[rajgene]

badcell, on Jan 27 2005, 09:42 AM, said:

Quote

how much DNA can you get from one miniprep? sometimes, i got very low concentration

I usually get around 20-40 micrograms DNA from a 3-5 ml overnight culture, using qiagen columns or something of the sort. The quantity of DNA will vary depending if your plasmid is low-copy or high-copy. One good thing is not to overload the column, use 3-5 ml for a high-copy plasmid, or 10 ml for a low-copy. This is a case of less is more. Cheers!

Hey Bad Cell the cloning master,
do u have ne idea of the percentage of false positives yeilded after transformation with and without rest.digestion after ligation? how many  colonies shld i pick for colony pcr for confirming the clone on rest. digestion after ligation
Thx man!!

[badcell]

Quote

do u have ne idea of the percentage of false positives yeilded after transformation with and without rest.digestion after ligation? how many colonies shld i pick for colony pcr for confirming the clone on rest. digestion after ligation

Hi, Rajgene. You can do a control ligation with vector alone, digest it and transform it in parallel with the good one with the insert. If everything has gone alright you should get around 3-10 times more colonies in the ligation with the insert than in the control one. In that case you can pick 2-3 colonies, and if you are in luck they will all be good. A month ago I got only 3 colonies in the ligation plate and none in the vector alone plate. I picked the three colonies, and alll three of them were good  

Quote

Thx man!! 

By the way, I'm a woman
Cheers!

[rajgene]

badcell, on Jan 28 2005, 08:03 AM, said:

Quote

do u have ne idea of the percentage of false positives yeilded after transformation with and without rest.digestion after ligation? how many colonies shld i pick for colony pcr for confirming the clone on rest. digestion after ligation

Hi, Rajgene. You can do a control ligation with vector alone, digest it and transform it in parallel with the good one with the insert. If everything has gone alright you should get around 3-10 times more colonies in the ligation with the insert than in the control one. In that case you can pick 2-3 colonies, and if you are in luck they will all be good. A month ago I got only 3 colonies in the ligation plate and none in the vector alone plate. I picked the three colonies, and alll three of them were good  

Quote

Thx man!! 

By the way, I'm a woman
Cheers!

good Badcell. thx for ur thoughts. btw i am using roche Rapid DNA ligation kit. it says heat inactivation of ligase will decrease transformation efficiency.
so my Q is how do u go abt digestion after ligation. do i have to heat kill ligase before i put my enzyme or i can directly perform on the mixture.
please let me know some details on how to go abt it.
thx Lady.

[george@CASE]

cool control, badcell!!   I will try this next time i do a ligation.

Doing a background control ligation is always important. Never do a ligation without negative controls, especially if you yourself cut the receiving vector.

[badcell]

Quote

. btw i am using roche Rapid DNA ligation kit. it says heat inactivation of ligase will decrease transformation efficiency.
so my Q is how do u go abt digestion after ligation. do i have to heat kill ligase before i put my enzyme or i can directly perform on the mixture.

Mm I didn't knew that. I'm using the Invitrogen T4 ligase and it doesn't say anything about heat inactivation. That's interesting. I'll try that next time, 'cause I usually inactivate it.

When I digest after ligation what I do is drop dialisis the ligation, you know, using Millipore membrane filters, for half-an-hour, and then add the appropriate RE buffer and the enzyme. I usually inactivate the ligase @ 65ºC for 10 min, but maybe this is not necessary, as changing the buffer and incubating at 37ºC may decrease a lot its activity. T4 ligase is unstable at high temperatures, and salt inhibits it. Cheers!

[rajgene]

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

[badcell]

Hi! Glad it went so well, rajgene
Next time I'll be sure to try without deactivating the ligase. Cheers!

[slapolla]

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.

Badcell-
that is indeed a great trick and would like to use it..but how do you know how much enzyme to add to the ligation..do you just base it on how much DNA you used in the ligation? And waht about matching enzyme buffer rxn conditions?

Slapolla

[badcell]

Quote

how do you know how much enzyme to add to the ligation..do you just base it on how much DNA you used in the ligation? And waht about matching enzyme buffer rxn conditions?

Hi slapolla
I don't really calculate the quantity of ER I add depending on the DNA used. I usually add 0.5 ul of ER and incubate for several hours (2-3 h, although sometimes I leave it o/n for convenience). This is more than enough, as your DNA would be on the ng range. Probably 1h of incubation will do it.
As for the buffer conditions, I sometimes dialyze the ligation for half an hour using spot dialysis filters from Millipore, and then I add the corresponding ER buffer and the enzyme. But I've also tried it without dialysis and it works. You can add directly the 10Xbuffer to the ligation mix, and the ER... and cross your fingers. For me this little trick really works.
Good luck