I am trying to do a routine cloning with GFP vector, 5500bp.
I need to double digest it with SalI and BglII.
I tried double digestion in NEB3.1, individual digest with each enzyme for 1hr then combine for another 1 hr digest and I also tried to add SalI first for 1hr then add BglII for an extra 1hr since some people suggest SalI has lower efficiency.
My testing gel may still look normal while when I am running all samples for gel purification it completely became smears. The condition I set is 80V x 70min on 1% gel.
Please help me with the problem! I am not sure if this is star activity or my DNA degraded? May this be related to the running buffer since it has been used for running many gels? If my DNA degraded, what might be the cause, as I did nothing but Qiagen purified it.
(The first picture is testing gel with undigested vector as control, the other bands are my digested DNA with different methods;
the second pic is my gel purification, which is totally smears)
Thank you David for your reply!
I will try it with longer incubation period. But still, I am curious about what makes my DNA smear-like? They appeared normal when I performed testing gel at 130V x 25min but came to a complete smear on my extraction gel ran at 70V x 70min. Is it purely due to incomplete digestion?
I will try today with also refreshed TAE running buffer and take a new NEB buffer3.1 for digestion. The chance for vector being contaminated is small.
The method I suggest that you can use BglII first, one hour later ,purification,and then use SalI cut for longer,as your method use Sall cut one hour and then add BglII looks like normal,but in digestion buffers there has BSA,one help thing,for a long time,it will gone or used that may not working for the next,
The condition that makes your DNA smear-like ,you can make a new gel,chang the TAE,(from your picture,your marker looks bad also),normally ,use 130Vx25 or 70Vx70 is not effect your test (if any doubt you can use another sample to test).
How clean is your DNA? Is it possible that it may have some nuclease contamination?
If your DNA is relatively impure, you may benefit from heating the DNA alone (in TE) at 75-80ºC for 5-10min and quickly chill in ice before beginning the restriction digestion. It will achieve two goals, kill many of the contaminating nucleases and relax the supercoiled DNA to make it a better substrate for your enzymes.
Best,
Thank you Fuxing for your reply. Indeed, I am trying to refresh all the solution and buffer and modify my electrophoresis setting. Apparently 70V is insufficient in my case.
It worth trying to purify the reaction after the first enzyme digestion, I will make it a try if necessary.
Thank you Tausif for your suggestions! My DNAs are routinely purified from Qiagen Mini Prep. I saw the testing gel as well as all previous gels showed no signs of extensive DNA degradation and I believe the gel on the 2nd picture is an isolated case.
Your method of heating DNA is very charming since one of my enzyme - SalI is very low yield when digesting supercoiled DNA. I believe this will help!
Best,
It seems you solved your problem by cleaning your buffers.
If the initial digestions show normal bands, but the DNA you get after gel purification shows a smear, the DNA degradation likely has happend in a step after the digestion. Thus, during purification, or possibly with the water / TE buffer you used to elute your DNA after cleaning.
One other possibility (like Niu fu Xing suggests); the buffer in your gel tank might be contaminated. If your lab re-uses the same buffer for running gels for a long time, DNAses can cause the DNA to become degraded while running the gel. We have seen that happen in our lab. Create a habit of regularly cleaning your gel tank and buffers.
I wonder if you have stopped the digestion before proceeding to the next step or not?
Thank you for your reply Arend! Indeed, I modified my experiment this way:
1. In 50 ul reaction, digest with 30Unit SalI for 3hr then add 15U BglII for an extra 1hr.
2. The vector is about 5500bp thus I run a 36cm-long 0.7% Gel and ran at 125V x 2.5hr.
3. I refreshed all the buffers like you indicated.
The results are good on the gel while minor shift of the band still exist. For my transformation, though, still I have several colonies grew up in my negative control. Thank you!
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