Hi Everyone, I am a perspective mater's student at UCSD. I been working on cloning and sub-cloning recently, I have ran into some issues; therefore, I just wish to see if there's any good trouble shooting tips that I can try.
Background:
I successfully cloned a 2kb gene encoding a yeast kinase into the pBluescript II KS (+) vector with a PCR-based cloning using the restriction enzymes SpeI and ClaI. The size of the final product is about 5kb. The final product is transformed into DH5α competent cells. The integrity of the insert was confirmed by sequence.
Current Experiment:
Now I am trying to sub-clone the insert from these pBluescript + Insert plasmid into pRS423 and pRS424 vectors with SpeI and ClaI. I normally set up a 20ul reaction with 2ug of DNA, 10 units of each enzymes (Total enzyme volume = 2ul), and final concentration of 1x cutsmart buffer for both the pBluescript + Insert and the vectors. The reaction is incubated at 37 degrees for 4 hours, and the enzymes are inactivated at 80 degrees for 20mins.
However, when I ran the product on gels, the "digested" pBluescript + Insert only gives me a strong band at 5kb when I am expecting two distinct band, one at 2kb (Insert) and the other at 3kb (pBluescript). For the pRS423 and 424 vectors, I often see a strong band at 6kb and a faint band a little bit below 3kb (I obtain a similar pattern when these vector were digested by either ClaI or SpeI alone)
Interpretation and Troubleshooting:
Based on the gel I got, I suspected that one of the enzymes in the double digestion failed to cut, resulting in band at 5kb for the pBluescript+Insert and bands at around 6kb for the pRS vectors. I reviewed the two restriction enzyme I have chosen for the sub-cloning. I noticed that ClaI is sensitive to supercoiled plasmid DNA, and ClaI activity is also blocked by dam methylation. I double checked the resource on dam methylation provided by NEB (https://www.neb.com/tools-and-resources/usage-guidelines/dam-and-dcm-methylases-of-e-coli) to made sure that ClaI is not blocked by dam methylation on my.
I also suspected that ClaI might also have trouble cutting the supercoliled DNA plasmid. Therefore, I modified the protocol based on my mentor's suggestion. I will add 10 units of SpeI into the reaction and allow it to incubate at 37 degrees. After one hour, I will add 10 units of ClaI into the same reaction and allow it to incubate at 37 degrees for another three hours, the enzymes are then inactivated by heat at 80 degrees. The rationale behind this modification was that if ClaI truly have trouble with supercoiled plasmid, maybe the first hour of incubation with SpeI alone will allow the plasmid to relax so ClaI can cut. However, even with this method, I still can't get the two bands (2kb and 3kb) I expected from pBluescript+ Insert.
I was wondering if you can help me with troubleshooting the subcloning? Maybe something I overlooked in the process or something I could do to improve the double digestion reaction? Thank you in advance. If there's any part of this post is unclear, I will be more than happy to provide additional information.
Generally your cloning looks good some points below:
1- during your sequencing confirmation did you confirm that the restriction sites are still intact?
2- You have too much glycerol in the reaction which might be interfering with the digestion I suggest you increase the reaction volume to 50ul.
3-One of you enzymes might have gone bad, try setting up single restriction to confirm. In case of claI if you believe supercoiling to be an issue then use a different enzyme that you know work (ask around in the lab for someone who recently digested with whatever enzyme).
4-Do an overnight digestion that might solve your issue.
Hello there,
for me it sounds like the restriction digest is very inefficient.
If you confirmed that the digest should work in theory (checked via sequencing, also found that ClaI is blocked by cpg https://nebcloner.neb.com/#!/redigest if this is of any concern), you could try to speed up the kinetics since you "only" incubate for 4 hours (i usually do this over night). Here i can recommend a protocol which lets you perform the digestion within 10 min, alternatevly to incubating over night (see article attached).
In short, i microwave the reaction mixture at max output power (800W) for 10 sec, and let it relax for 2 min. Repeat the step 5 times and you´re done.
Best of luck,
David
Article Microwave-mediated Enzymatic Modifications of DNA.
As a general recommandation always start with one enzyme and check that the plasmid is linearized if not you have trouble with your digestion mixture either DNA trouble or most of the time enzyme problem. In the good old times I remember having so much trouble with ClaI enzyme.
So take your plasmids and do one cut at a time and in separate digestions. you should be able to see which enzyme gives you trouble. Also check teh NEB Biolabs table for survival of restriction enzymes to make sure that you're in the best conditions.
Remember that some enzymes like BamHI for example doesn't like to cut in the common restriction buffer and prefers it's own buffer. Hope it helps
Hi Hanna Alalam Thank you for the suggestion. I double checked the sequence data and I think the restriction site is intact. That was something definitely worth paying attention to. I just want to check in with you to see that for the ON incubation, we would just leave the reaction at 37 degrees and perform the heat inactivation the next day?
Thanks for the reply Jean-Pierre Dangy , it's comforting to know someone else also had trouble with ClaI haha. I just did a single digestion with either SpeI and ClaI with the pRS vector backbones (~6kb). The digestion gave me a strong band at around 6kb and a very weak band right above 3kb. I thought this is an indication that the enzyme still works as intended. I plan to repeat this with the pBluescript + Insert plasmid since I concentrated this plasmid by EtOH precipitation after miniprep. If the digestion does not work, I think I might have DNA problem.
Perform the reaction in a total volume of 50ul in a pcr tube setup the PCR machine to incubate at 37 overnight (remember to set the the lid to 40c to prevent evaporation). Since you are performing gel purification heat inactivation is not necessary
Make sure that you dry well your DNA pellet as contaminating ETOH is very potent in blocking restriction enzymes. As for your single digest you expect only a single band with one enzyme isn't it ?
Jean-Pierre Dangy Yes! I think the expected result of the single digestion should be one band. I think the weak band was uncut plasmid from inefficient digestion. Since the strong band is so much stronger than the weak band. I thought the digestion worked. Would this be a correct interpretation. By the way thank you for confirming the effect of EtOH on restriction enzymes. I feel a little more confident about my trouble shoot solutions.
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