Mysterious Band in NdeI/BamHI Double Digest - (Sep/30/2008 )
It's my first time posting, but I am an avid reader and a big fan of the forums on this site - I have found them very useful.
Anyway, here's my problem in a nutshell:
I did a double digestion of ~2.5µg pET-11a with NdeI and BamHI (both from NEB), followed by a dephosphorylation with Antarctic Phosphatase. Upon running a gel, I noticed that there were two distinct bands in the doulbe-digest (see picture).
Let me be clear about what all the lanes are:
Lane 1 - control digest with NdeI (~200ng vector)
Lane 2 - control digest with BamHI (~200ng vector)
Lane 3 - uncut pET 11a (~200ng vector)
Lane 4 - log 2 ladder (marker)
Lane 5 - empty
Lane 6 - pET 11a double digest after dephosphorylation (~2.5µg vector)
Here is my detailed experimental protocol for the double digest:
01. Added 19µL of 127ng/uL pET11a to 10µL milliQ water
02. Added 4µL 10X Buffer 3 (NEB)
03. Added 4µL 10X BSA (NEB)
04. Added 1µL NdeI + 1µL BamHI (total volume now 39µL)
05. Incubated 1.0 hr at 37°C
06. Added 1µL NdeI + 1µL BamHI (total volume now 41µL)
07. Incubated 1.0 hr at 37°C
08. Added 30µL milliQ water
09. Added 3µL 10X Buffer 3
10. Added 3µL 10X BSA
11. Added 1µL NdeI + 1µL BamHI (total volume now 79µL)
12. Incubated 1.0 hr at 37°C
13. Added 9µL 10X Antarctic Phosphatase Buffer (NEB)
14. Added 1µL Antarctic Phosphatase (NEB) (total volume now 89µL)
15. Incubated 0.5 hr at 37°C
16. Added 1µL Antarctic Phosphatase (NEB) (total volume now 90µL)
17. Incubated 0.5 hr at 37°C
18. Heat inactivated Antarctic Phosphatase, 65°C for 5min
19. Added 10µL 10X DNA gel loading buffer (total volume now 100µL)
20. Froze at -20°C, stored overnight
21. Poured 50mL, 1% agarose gel with EtBr, ran at 200V for 40min
In total this is a 4-hour incubation with new digestion enzyme added about every hour. The increases in volume are used to keep the total glycerol concentration from the enzymes under 10% (v/v).
My thoughts are that dephosphorylation may have caused another band to form in my double digest, although I am not sure how this is possible. With the combination of buffers present in my mixture, it is hard for me to to decide what it going on. I'm just curious if anyone has seen anything like this before.
My plans are to gel extract both bands from the double digest and try test ligations with each of them to see which bands undergo successful insertion. After sequencing I should have some answers... but until then, would anyone care to speculate on what might've happened?
I am currently dealing with NdeI and BamHI too.
My problem is exactly same as yours. Single digestion with either NdeI and BamHI showed almost complete digestion. However once i perform double digestion with both of them, the result is same as yours, which is incomplete digestion.
You can try to do sequential digestion. Means you digest your plasmid with NdeI first(buffer 4), then purify, after that digest again with BamHI(buffer 3). I have tried to perform d.digestion for 2 hours, 6 hours and even overnight but no improvement is observed.
I did not put in phosphatase, so i think it is not the source of problem.
Just as a note, i am using NEB enzyme and i dealed with pET family(pET14b). This are two similarities that both of us involved.
This is an odd problem. In pET14b the BamHI and NdeI sites are 6bp apart. NdeI requires 8bp to cut, thus it is not possible to conduct a double digest with this vector. BamHI is also a move active enzyme and comes in a more concentrated form.
In pET11a and pET11b, the BamHI and NdeI sites are 31bp apart, separated by the T7 Tag. So there should be enough bp skirting the NdeI site for the enzyme to cut this site efficiently
Could you confirm that you do have pET11b rather then any other version of the pET vector?
If a double digest is done, I would suggest increasing the digest volume to 100ul and leave to digest overnight.
If you do a sequential digest start with NdeI, confirm that the digest is complete and proceed with BamHI.
Personally I also don’t think continuously adding enzyme every hour is useful and is somewhat wasteful. The enzymes will remain active longer then that. And it is time consuming on your part.
I think it is better to keep the total enzyme concentration below 5%v/v rather than 10%v/v. It is rather high and if any enzyme sticks onto your pipette tip, can easily tip it over 10% where enzyme inhibition (from glycerol) and star activity starts appearing.
So matthew m, maybe you can try to do sequential digestion.
Sorry, i should make it clearer.
Actually my vector is recombinant vector with insertion (~1kb) in between NdeI and BamHI.
When i digest my insertion out with NdeI and BamHI, the band is very fade. partial digested plasmid can be observed as with matthew m gel picture.
When i confirm the plasmid backbone, for example NdeI+EcoRI and BamHI+EcoRI, the band is very bright with complete digestion of plasmid.
I have deal with digestion for quite a long time but it seems that this couple (NdeI+BamHI double digestion) still do not want to befriend with me
when they divorce, they do.
I have confirmed that the vector is pET 11a by sequencing.
It seems that the consensus among the replies is that my problem is incomplete digestion. However, the band for uncut vector and the upper band in my double digest are in different places. Thus, if the problem is incomplete digestion, how come the bands associated with uncut vector are not present in the double digest? Could there be another possible explanation?
My only hesitation about doing a sequential digestion is that it most likely requires two gel extractions. I get very low yields with gel extraction (Qiagen Kit) - so I try to avoid it whenever possible. Also, the post-doc in our lab who gave me this protocol usually prepares the vector for our experiments and it always works for him. I have found that his doubly digested vector is the most reliably prepared in the whole lab, which is why I wanted to try his method. The presence of this extra band has left us all perplexed though.
By the way, thank you to everyone for your replies and inputs
That is one question answered.
True, but the band for the linearised DNA in the double digest did not run in the same place with either the linearised DNA from the single digest. That band isn't even straight. So I would say the running of the gel has not be perfect... the band at the end of the gel look to be running at an angle.
Possibly, however finding the why of this mystery is not important, only the how to get around it. As my boss would say, "Solving ligation mysteries won't get you papers. Just get the plasmid built."
Gel extraction is not required. Start with NdeI in buffer 2. Once the DNA is digested add BamHI. BamHI is a good enzyme which cuts well.
Once you have melted the agarose gel in QG buffer, pass this solution three times through the column, this gives the silicon matrix more time and opportunity to bind to your DNA. And when you add the elution buffer, use elution buffer that has been warmed up to 68 Celsius. Also leave the elution buffer on the column for about 2 minutes. Also elute the DNA from the column in two round... say you want 50ul ... first elute with 20ul, then in a second round elute with 30ul buffer. This should give improved yields.
Thank you so much for your help! I think that that advice from your boss might've just changed my life
The tips you gave on gel extraction were excellent, my yields are a lot higher now. Thank you!
And for a happy ending, I repeated the digestion with a slightly different protocol to get the following gel:
01. mix up 40µL of miniprepped DNA with: 6µL Buffer 3, 6µL 10X BSA, 2µL milliQ water
02. add 1µL NdeI + 1µL BamHI for a total volume of 56µL
03. incubate at 37°C, adding 1µL of each enzyme every hour until you reach 60µL total
04. after 3 hrs total, add: 11µL milliQ water, 8µL AP buffer, 1µL Antarctic Phosphatase
05. incubate at 37°C for 15 min
06. heat-inactivate at 65°C for 5 min
07. 10X DNA gel loading buffer
08. run on gel, gel-extract
*(if you have less than 40µL DNA, dilute with milliQ water to that volume)
*(make sure to wipe the pipette tips with a KimWipe to avoid adding extra enzyme soln.)
Maybe I did add to much Antarctic Phosphatase the first time, but who cares right? It worked this time!