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double restriction digestion - no ligation (Jun/18/2005 )

Hi! All
there's a query regarding the double digestion of a vector of 3.4kb size, I am digesting it with HindIII and BamHI but getting either partial digestion or some times not at all. The two enzymes are from MBI Fermentas where there is a star activity when 1X Tango buffer is being used but for 2X buffer there is 50-100% activity for both the enzymes when opting for double digestion. taking 2.5u of BamHI and 3.5u of HindIII.
I am not getting gud results by this method, now i am trying to do it sequentially. first digesting it with HindIII and then cleaning it up, and further moving to secound digestion with BamHI.Here can somebody guide me with which reaction conditions to opt for.
Thanx sad.gif

-tweety-

Hi tweety,

I used to double digest with NcoI and BglII and I just simply used the buffer recommended for double digestion with these two enzymes and got no problem.

I think first you should check out that your vector is free from any contaminants that can inhibit the enzymes' activities.

If you try to digest in-serial and it do not work, I guess that there is inhibitor(s) in your reaction solution.

-dtle-

QUOTE (dtle @ Jun 18 2005, 02:07 AM)
Hi tweety,

I used to double digest with NcoI and BglII and I just simply used the buffer recommended for double digestion with these two enzymes and got no problem.

I think first you should check out that your vector is free from any contaminants that can inhibit the enzymes' activities.

If you try to digest in-serial and it do not work, I guess that there is inhibitor(s) in your reaction solution.


Hi dtle,
if there is any kind of contaminant, hw to get rid of it firstly? Secondly, i m using plasmid purification kit from Qiagen n i think it is quite reliable. wat do u say about the chances of getting contaminants in my plasmid. regarding my insert (PCR product) i m again going gel extraction using the kit from Qiagen. Please give me some clue......... i m getting nuts. It is nw complete one month i m not getting ligation.
ohmy.gif

-tweety-

hi tweety,

I believe the Qiagen KIT works fine. Double check the purification of your insert. The contamination could come from the gel, electrophoresis buffer, loading buffer.

My friend used to have similar problem this, and finally, we figured out the inhibitors came from the loading buffer.

-dtle-

QUOTE (dtle @ Jun 20 2005, 05:45 AM)
hi tweety,

I believe the Qiagen KIT works fine. Double check the purification of your insert. The contamination could come from the gel, electrophoresis buffer, loading buffer.

My friend used to have similar problem this, and finally, we figured out the inhibitors came from the loading buffer.


hi dtle and everybody else around!
i am not getting the digestion, i have cross checked that enzymes are not the problem. i hv used the enzymes from both MBI fermentas as well as NEB. Regarding my vector wen i run it on gel, i m able to see the 3 defined band without ny smearing. the quality of the vector luks gud and i m purifying the plasmid using plasmid purification mini kit. The concentration of the vector on gel analysis is 100ng/ul.
for digestion the reax conditions i m using for 20ul reax mix are as follows
plasmid = 10ul
buffer= 2ul
hindIIIenzyme = 1ul (20u/ul NEB)
BamHI=1ul (20u/ul NEB)
deionized water= 6ul
I have tried to do even single digestion, but all in vain...........
on gel analysis i m seeing the vector as it is. please help. ITs URGENT.
thanx

-tweety-

Hi tweety,

First of all have you checked your digests at each stage when you do it sequentially. After digestion with HIndIII do you linearise? If you cut with BamH1 do you linearise?

The only thing I can think of is that there is something wrong with one of the restriction sites in the vector. Have these sites been PCR amplified, even if they are within pirmer regions I've found mistakes in commercially synthesised primers before. Also is there any methylation sites which could interfere with the restriction sites, you may have to grow the plasmid in a different strain of bugs.

All I can think of at the moment. But a couple of things to check.

Can you use two other enzymes?

Cheers,

Scott

-Scott-

hi
you've mentionned in your first post that with 2X buffer things were good. Hence 2µl of buffer seems to be 1X final...
Moreover, i assume that enzymes are in 50% glycerol?
By adding 1µl of each enzyle, you are at the inhibiting concentration of glycerol in your final.

I was wondering: if you do a sequential digestion, 1µg is quite few. I would suggest to do a sequential digestion in 50µl minimum 5why not 100µl for the first, and let say 50µl for the second one?
I would do a first digestion with HindIII. Not good arguments, but i use regulary BamHI overnight and never saw star activity. Hence youcand do 4h with Hind III precipitate wash and do your second digest overnight.

But scott is right. You should checkat each stage the progression of your digestion...

Hope that helps...
Fred

-fred_33-