Restriction Digest with pGEMT for TA cloning - (Mar/27/2017 )
Am gonna re-transform using the 3 sample DNA that showed my insert on the gel. I will then prepare glycerol stock using positive clones from the transformation i am going to do so that i dont have to "re-transform" again. I have ran out of Plasmid Miniprep kit, so i will have to use the traditional alkaline lysis method, which i have not done before, hope and pray it works out. I will keep you informed as i appreciate and value your unreserved knowledge you have been sharing thus far!
No problem, with the advice, that's what we are here for...
The alkaline lysis works well, I've used it routinely in the past. Just pay strict attention to the times for the initial lysis and neturalization steps, and make sure you don't overdry the DNA at the end.
Actually, the kits for plasmid preps are all based on the alkaline lysis method, so the steps are essentially the same, other than that the kits generally use a filter to get trap the DNA, rather than spinning it down.
Things seem to have gone from bad to worse for me. I started from the beginning by doing pcr, which works every single time, BUT the concentration after gel purification (GenJet Thermo Scientific kit) is very low (25ng/ul). I try to increase the wells by taping the gel comb, which i did the first time and worked, but this time, when i tape, i dont get a very good desired product run. What can you recommend i do to have a high DNA concentration following Gel purification or prior to? Really frustrating! Do you know about "nested PCR"? How does one go about it? I am running out of my DNA and would rather amplify ones and use that pcr product as a template. 
Gel purification always gives low concentrations, 10-25 ng/ul is normal for an input in excess of 1 ug (also note that 25 ng x 50 ul = total amount). It should be a last step in most procedures where you need pure DNA. It may be that you don't need to do a gel purification step. I would only do one if you have many bands in your PCR, in which case you should have optimized the PCR anyway... If the gel extraction is the last step before ligation - you actually need very very little DNA for a ligation, I typically use 20 ng or less total DNA in a 20 ul reaction (NEB recommends 1-10 ug/ml, in other words 1-10 ng/ul total DNA). Ideally you would use molar ratios of the insert:vector so that you optimize the chance of ligating the insert into the vector. Calculate these using the following formula - note that the example uses unrealistic amounts of DNA!:
((ng vector)x(kb size of insert))/(kb size of vector)) x (molar ratio of (insert/vector)) = (ng insert)
example: 500bp insert to be ligated with 100ng of 3.0kb vector in a 3:1 ratio
((100ng vector)x(0.5 kb of insert))/(3.0 kb vector)) x (3/1)) = (50 ng insert)
If you are amplifying the DNA with restriction sites on your primers, I would just purify the DNA from the reaction, then digest (you can do double digest for Nde and Xho, buffer 4 from NEB - if you are not using NEB, check the guidelines from your supplier). After this step you should only be getting rid of tiny fragments of less than 20 bp, which should not come through any purification procedure (they are too small to spin down easily for precipitation procedures, and too small to bind to columns efficiently). You should then be able to do a ligation with the end result, no need for gel extraction.
Nested PCR is where you take a second set of primers that are inside the amplicon of a first PCR and use those to re-amplify the product. You can not use this easily to get full length product! It also increases dramatically the chance of there being an error in the final sequence. I would avoid if possible.
Back to restriction enzyme digestion. I did a restriction enzyme digest as follows (50ul total volume): 39ul dH20, 3ul DNA, 5ul O Buffer, 1ul NdeI and 2ul XhoI. The digestion was carried out for 3 hours at 37oC in a heating block.
I have attached the gel of 7 samples of which 6 had all the reagents including enzymes and the first one had no enzyme. My insert is 1197bp and the vector is 3000bp (pGEMT).
One of the enzyme expired in 2013 and the other is meant to expire in 2019. I think the enzyme are not cutting because if you remember from my post above, i said i once cut with EcoRI and it worked (for 1 hour).
Having said that, i have left my digestion reaction at 37oC for over night. I will then run a gel to see if the overnight reaction was enough for the enzyme to cut.
Some more information that i think might be helpful in you giving me a detailed answer, here are the Nanodrop results after Plasmid Miniprep in ng/ul:
Sample: 1kb ladder, 2 (75.8), 3 (45.2), 4 (66.5), 5 (70.8), 6 (45.8), 7 (45.8)
If you have any suggestions, please help a new scientist out.
I have a codon-optimized gene cloned into pUC57 with AvrII and XmnI overhangs. I want to subclone the gene into a yeast expression vector (pKOV410). The size of my gene is 1429 bp and the expression vector is 6352 bp. The overhangs are close to each other on the vector. I did a doble digest on the pUC57 to release my gene, ran a gel and extract my gene of interest using Promega gel extraction kit with a final elution volume of 50 ul. I get 20ng/ul concentration of my gene. I also did a double digest of the pKOV410, run and extract the linearised vector. The nanodrop concetration of the vector is 45ng/ul. I am using NEB ligation kit. I tried ligation at different environment (3hr RT, 16hr O/N, 4 degrees O/N). I take at least 3-5 ul of my ligation into 50ul JM109 competent cells which we just bought. I do a control with uncut vector and i get hundreds to thousands of colonies. The ligation kit is new and i made aliquots of the buffer the day i received the kit. The enzymes are from NEB & they recommend digestion for 5-15min, but it seams not to be enough, so i went for 1-3 hours, that works well. But i still dont get a single colony. One of the overhangs is blunt & the other is sticky. I have not tried sequential digest yet. The antibiotic resistance for the expression vector is Kanamycin. I have tried different ligation ratios (1-20) but NO colonies.
ANYONE WITH SUGGESTIONS THAT CAN HELP ME GET PASS THIS STEP?
In response to your previous post:
I would say that you have one enzyme that is cutting completely - this is good, it's what you want to happen. I say this because plasmid preps usually contain multiple forms of the plasmid in different energy configurations, such as supercoiled, circular, linear, nicked etc. Your prep appears to have most of the DNA in one form, this is most likely circular (it's running small), which means that you did just fine in your prep, but you should pay strict attention to the time for alkaline lysis. If you do this you will get some super-coiled out too. Your concentrations are a little low, but not too bad for a beginner.
Your reaction conditions are good - you have at least a 2x excess of enzyme over DNA.
However, the other enzyme is not cutting - most likely this is the XhoI which works less well in buffer O. One good way to get around this is to do sequential digests - do one digest, clean up the DNA by precipitation, then do the next digest in the recommended buffer for that enzyme. To do this it is a good idea to start with a lot of DNA (maybe a ug) and work from there. Don't scale up the volumes for the digest, just make multiple tubes of the digest reaction.
If your enzymes have been stored correctly and kept cold (i.e. only taken out of the freezer just as you are adding it to your reaction) then they should last more or less forever. I have (admittedly 5 years ago) used enzymes that expired in the 1990's with no problems.
Which ladder are you using for your gels? Knowing the band sizes is half the battle in working out what might be going wrong.
Mashudu on Fri May 4 13:00:17 2018 said:
I have a codon-optimized gene cloned into pUC57 with AvrII and XmnI overhangs. I want to subclone the gene into a yeast expression vector (pKOV410). The size of my gene is 1429 bp and the expression vector is 6352 bp. The overhangs are close to each other on the vector. I did a doble digest on the pUC57 to release my gene, ran a gel and extract my gene of interest using Promega gel extraction kit with a final elution volume of 50 ul. I get 20ng/ul concentration of my gene. I also did a double digest of the pKOV410, run and extract the linearised vector. The nanodrop concetration of the vector is 45ng/ul. I am using NEB ligation kit. I tried ligation at different environment (3hr RT, 16hr O/N, 4 degrees O/N). I take at least 3-5 ul of my ligation into 50ul JM109 competent cells which we just bought. I do a control with uncut vector and i get hundreds to thousands of colonies. The ligation kit is new and i made aliquots of the buffer the day i received the kit. The enzymes are from NEB & they recommend digestion for 5-15min, but it seams not to be enough, so i went for 1-3 hours, that works well. But i still dont get a single colony. One of the overhangs is blunt & the other is sticky. I have not tried sequential digest yet. The antibiotic resistance for the expression vector is Kanamycin. I have tried different ligation ratios (1-20) but NO colonies.
ANYONE WITH SUGGESTIONS THAT CAN HELP ME GET PASS THIS STEP?
OK. First things first - you know your digest worked on pUC57, but did it work on the pKOV410?
Are you growing the pUC57 yourself or did you get it straight from a vendor? If from the vendor, check the sequence, perhaps it doesn't grow well.
Make sure you are growing at 30C for the pKOV under Chloramphenicol selection (At least for pKOV strains according to Addgene - but check your product sheet)
Blunt ligations are inefficient, overnight at room-temp should work. If you are still struggling, try adding some PEG8000 as a crowding reagent.
Try adding some more ATP to your reaction - it easily degrades even when stored properly.
It is counter-intuitive, but ligation reaction components can be inhibitory to transformation - try transforming 0.5, 1, 2, 3 ul of the reaction and plating.
Upon receiving the pUC57, I transformed and grew the pUC57 and made glycerol stocks so that i do not run out of DNA. I am using the glycerol stocks in my experiments. I have been growing pKOV at 37C. What about the fact that the two ends are close to each other? Would you suggest that i do a sequential digest?
It might be a problem, but so long as they are more than about 3 bp apart, it should be fine, so long as cutting with one doesn't disrupt the recognition sequence for the other. I see that XmnI has a large recognition site, so if you were to do a double digest, I would do that one first.