15 replies to this topic

[zarqoo]

I am trying to digest the vector using BamHI and SacI. Sequential digestion is recommended for this so im going to do SacI then BamHI after.

(I tried double digestion with BamHI and SacI but no success.)
I tried sequential digestion by digest with SacI first (Neb buffer4 2 ul, 10x BSA 2 ul, SacI 1ul, plasmid 3.5 ul and water 11.5 ul) After incubation at 37 c for 3 hour I heat inactivate SacI in 65 c 20 min.
After the first digestion (and purification) I end up with a 20ul DNA in water.
Then I do second digestion with BamHI (Neb buffer3 2 ul, 10x BSA 2 ul, BamHI 1ul, first digest product 3.5 ul and water 11.5 ul)

In the end I cannot get the expected product , I don't know why?
Can anyone suggest me the appropriate protocol for doing double digestion with BamHI and SacI ?

Thanks,
Zarqoo (Thailand)

[leelee]

What do you mean can't get the expected product? Is it the wrong size? No DNA present at all?

If I am understanding you correctly, you start with 3.5ul plasmid- which by the process of your digest and purification is diluted into 20ul of water, of which you only use 3.5ul for the 2nd digest. Perhaps your DNA is too dilute to see by AGE (is this how you are checking it??) by the time the second digest is done??

[zarqoo]

leelee, on Sep 23 2009, 08:13 AM, said:

What do you mean can't get the expected product? Is it the wrong size? No DNA present at all?

If I am understanding you correctly, you start with 3.5ul plasmid- which by the process of your digest and purification is diluted into 20ul of water, of which you only use 3.5ul for the 2nd digest. Perhaps your DNA is too dilute to see by AGE (is this how you are checking it??) by the time the second digest is done??

Thank you leelee

I mean that I cannot get the product, I think it cannot cut because the band is similar to uncut lane, and in this experiment I want 100 bp product but I cannot get any.

I agree with you that sometimes the DNA in 2nd cut is too dilute.
Can you suggest How much DNA should I add into the reaction? (also how much Buffer, 10xBSA, SacI )

Thanks

[leelee]

So what you are saying is that when you run your second digest out onto a gel, there is no difference between it and an uncut control?

For your double digest, I would do the following:

1. SacI digest (just as you have been)
2. Purify (you won't need to heat inactivate the enzyme if you are purifying, so you can remove that step)
3. Do BamHI digest, but use 15ul of your purified SacI digest instead of only 3.5ul
Your amount of enzyme, buffer and BSA seems fine to me.....

Are you using AGE conditions appropriate for resolving a 100bp band, and a 100bp diff between cut and uncut? It may be that you are actually getting what you want, but just can't see it clearly on your gel?

[bac]

Hi there!
Hope you solved the issue meanwhile. If not, I hope this helps.

Another option could be to get the HF versions of the two enzymes, which should work out well together in buffer 4.
You also may want to double check if you are not using the HF versions with the "previously advised buffers"

[stardust]

Hi,

are you trying to obtain the vector backbone after removing 100 bp or are you interested in the 100 bp fragment? When you say 3,5 µl DNA, how much is that (in µg) and how big is your plasmid in total?

Stardust

[zarqoo]

Thank you "leelee" "bac" "stardust" for your suggestion

stardust, on Sep 24 2009, 07:05 PM, said:

Hi,

are you trying to obtain the vector backbone after removing 100 bp or are you interested in the 100 bp fragment? When you say 3,5 µl DNA, how much is that (in µg) and how big is your plasmid in total?

Stardust

(I am interested in 100 bp fragment and after purification I obtain DNA around 100 ug/ul)

today I try to add 15 ul of DNA in the second digestion  but it did not work ( |||).
In the gel I can see the band of plasmid that similar to the band from cut with 1 enzyme (BamHI or SacI)
so I incubate them overnight but the result is like incubate 4 hour (can not get 100 bp fragment)

-->I also try to digest with BamHI befor SacI but the result is still not work (T_T)

[zarqoo]

bac, on Sep 24 2009, 03:25 AM, said:

Hi there!
Hope you solved the issue meanwhile. If not, I hope this helps.

Another option could be to get the HF versions of the two enzymes, which should work out well together in buffer 4.
You also may want to double check if you are not using the HF versions with the "previously advised buffers"

May be I should think about using HF version if I cannot digest with the existing enzyme. (T_T)

[lizzy]

Maybe the two enzymes work well and cut the 100bp fragment, but you cannot see it on gel. Can you tell us the amount of the digestion action you ran on the gel? Maybe the amount of 100bp fragment is  so small that you cannot see it on the gel.

Edited by lizzy, 24 September 2009 - 07:08 PM.

[zarqoo]

lizzy, on Sep 25 2009, 10:07 AM, said:

Maybe the two enzymes work well and cut the 100bp fragment, but you cannot see it on gel. Can you tell us the amount of the digestion action you ran on the gel? Maybe the amount of 100bp fragment is  so small that you cannot see it on the gel.

Thanks lizzy

I take 7 ul of the digestion action to ran on the gel.
From the gel result I can see only the plasmid band that similar to the lane that digested with 1 enzyme but different from lane that run the uncut plasmid and I cannot see 100 bp band.  

[stardust]

Sorry did you really mean 100 µg/µl? or did you mean 100 ng/µl? 100 µg/µl seems quite unrealistic to me...Lets say you have 100 ng/µl and you use 3.5 µl. that means you digest 350 ng. This is not enough to see the 100 bp fragment in my opinion. Try digesting up to 10 µg with 3 U BamHI per µg and then purify and then digest everything you get (!!) with 3 U SacI per µg DNA. see if this works. For the second digest, if you get 20 µl DNA, then perform the digest with all of it in a total volume of let say 30 µl and put all of it on the gel. You have to consider that lets say your palsmid is 3000 bp, then you have 350 ng. The 100 bp fragment therefore corresponds to only about 10 ng of your total 350 ng! that is not enough to see properly on the gel. If you digest 10 µg then to about 300 ng which you should be able to see and cut out the band.

Stardust

PS: could you attach a gel picture?

Edited by stardust, 25 September 2009 - 02:06 AM.

[zarqoo]

stardust, on Sep 25 2009, 05:05 PM, said:

Sorry did you really mean 100 µg/µl? or did you mean 100 ng/µl? 100 µg/µl seems quite unrealistic to me...Lets say you have 100 ng/µl and you use 3.5 µl. that means you digest 350 ng. This is not enough to see the 100 bp fragment in my opinion. Try digesting up to 10 µg with 3 U BamHI per µg and then purify and then digest everything you get (!!) with 3 U SacI per µg DNA. see if this works. For the second digest, if you get 20 µl DNA, then perform the digest with all of it in a total volume of let say 30 µl and put all of it on the gel. You have to consider that lets say your palsmid is 3000 bp, then you have 350 ng. The 100 bp fragment therefore corresponds to only about 10 ng of your total 350 ng! that is not enough to see properly on the gel. If you digest 10 µg then to about 300 ng which you should be able to see and cut out the band.

Stardust

PS: could you attach a gel picture?

Thank you stardust  
Your advise is very clear. I've never think about this point.
Now, I do not have gel picture. May be I will attach the picture on Monday.
Have a good week end. \(^o^)/

[lizzy]

I agree with stardust. I often digest 1ug vector containing about 200bp insert with 10U enzyme such as xhoI, xbaI, then run all the digestion reaction production on the gel. I can see the 200bp fragment clearly.

Edited by lizzy, 27 September 2009 - 11:53 PM.

[stardust]

Hi there!

Did it work?

Stardust

[atin]

Hi all!
I am also facing some problem using these two enzymes. I am trying to digest pQE30 vector with BamHI and SacI. When I digest about 500ng of my vector with BamHI (using ~6 units) in a 20ul reaction, even after 4 hours of digestion I am able to see the plasmid undigested on 0.8% agarose gel. The NEB catalog says that BamHI shows immediate activity, then why am I getting the uncut vector? (the enzyme is also new)
And I have been performing double digestion for quite a time but I am not getting a double digest. I have tried to digest sequentially as well as both enzymes together in the same reaction. But in all the cases I see the vector self-ligate upon ligation.

I need some help please!