blunt-cloning - all are vector self-ligation products? (Jul/11/2003 )
In my experience a vector to insert ratio of 1:8 or 1:10 works better for blunt end cloning. 1:3 is good for sticky ends (I know the paper that comes with the ligase says 1:3 for blunt end, but forget about that).
You also need to take into consideration the length of vector and insert according to this formula:
100ng vector\length of vector in bp = X ng\length of insert
Once you've solved it to X, just multiply X by 10 and you have the number of ng of insert you need to use for 100ng vector.
Also as someone already mentioned, use higher concentrated Ligase, but keep in mind that the glycerol concentration from the Ligase storage buffer should not exceed 5% in the ligation mix.
When you say that you have purified your vector properly, does that mean you've done it by gel-purification? If not you might still have uncut vector there. On the other hand, your neg. control seems to be ok.
The vecotr insert ration should be atleasr 1:3
Uh you do know that Taq tends to add overhanging As to the end of any PCR product? This can be a problem if you don't want extra nucleotides in your cloning site.
On the other hand you can use this ability to help with blunt end cloning. It's called T/A cloning, invitrogen sells pCDNA3.1 vectors that already have overhanging Ts. But if you need a specific vector you can make the overhanging Ts yourself. Basically you need to incubate the vector with dTTP and Taq as described here:
http://www.oup.co.uk/pdf/pas/9v1-7-14.pdf
Hope that helps
Way I did it (successfully):
- purify insert + vector with prefered method
- IF cutting with the same REs, mix plasmid + vector together and digest
- purify with prefered method
- add salt, dNTPs and Taq, incubate for a couple of hours
- clean up again
- add ligase + buffer etc and incubate o/n
- don't forget to pray between steps :-P
hi... do you have the protocol using Tag... I mean, dNTPs concentration, enzime units, DNA concentration, Temp, etc. I read it's a good alternative to klenow or S1 nuclease. Thanks
hi,
isnt it necessary to phosphorilate the pcr insert if the vector is dephosporilated? for pcr products are lack of phosphate in the 3' end, isnt it?
cheer
chandima
yes it works very well in our experiment,too.
and i add the PEG4000 for the ligation.
havent you tried inactivating CIAP with edta at around 75 degree C. also you can use sds with edta at a lower temperature. if you feel that still its not working try using SAP.
all the best and get back with the positive result soon
yes it works very well in our experiment,too.
and i add the PEG4000 for the ligation.
I just started the cloning work few month ago.
So I have many questions about cloning.
In this case why did you used PEG4000 for the ligation, and what properties did you use for it?
Personnally, I won't de-phos at all. Yes you'll get squillions of ligation positives but you can screen 50 - 100 very easily using single-colony gels (or blue/white selection even better of course if you have it). A single colony gel (if unfamiliar) is simply a blob of +ve colony lysed in SDS buffer (with a dye), spun and ran on a large agarose gel. An insert will cause the supercoiled plasmid to run slower than plasmid w/o insert. I've used this technique to detect a 50bp insert in a 6kb plasmid.
zing
Could you please email me a detailed protocol for single-colony gels? Thank you. (fangj1@mmc.org)
Hi,
I agree with the others that CIP is not efficient enough. I also use SAP for this purpose and recently I used Antarctic phosphatase, It is perfect bc the inactivation takes only 5 min!!!
Hope it helped
[quote name='popogirlxd' date='Jan 2 2005, 09:39 PM' post='9705']
[QUOTE].after several defrozing steps, ATP could be depeleted. I improved results adding ATP 10 mM to ligase reactions. If the buffer is not so old, aliquote it in small amounts, just to use once or twice
Hi, Enthusiast
I am doing blunt-end ligation too and also have the trouble to get the clony. You suggestion is very good and I will try it. I am eagerly to know that why the ATP could be dpeleted after using several times. Could you tell me the answer?
Thanks
[/quote]
As said above, aliquoting the ligase buffer as soon as you receive it is the way to go - I would avoid using an aliquot after more than 1-2 freeze-thaw cycles. Sounds wasteful, but you get a huge excess of buffer every time you buy ligase. As for ATP degradation, it's just simple chamical hydrolysis. The phosphate bonds in ATP are pretty labile, and each freeze-thaw cycle hydrolyses some more of the ATP to ADP (a major cause of grey hair when cloning...).
D.
your question is why those clones did not contain of inserted DNA as the control is totally negative, is it?
the reasons could be the insert DNA contaminated bacteria, whose can grow in antibiotic agar plates. if this is the reason, you will see bacteria grows on plates as you spread the insert DNA solution in antibiotic containing agar plate.
PEG is good for the ligation reaction. Proper PEG concentration is able to increase ligation efficience.
it is true the efficience of blunt end ligation reaction is relatively lower than sticky end ligation under the same condition. To increase the reaction efficience, you can adjust the properation of insert DNA and vector.
Ratio of inert DNA and vector is improtant. what ratio is best I do not know. case by case, the only way is try. On earth, it is experiemnt. this does not have criteria or rule that you can follow up.
do not wast time to prepare the new vector or dephosphorlation. In your case, the vector that you had done is good.
Dear Karen,
there is now a system available called staby cloning from Eurogentec. With this you get just clones if your insert is incorporated in the right direction, since you add a missing part of the selection marker to one of your primers. There are no re-ligations, no empty bacteria, because the bacteria are coding the selective poison by themselves. Maybe that might help you.
Cheers,
ophris