Restriction enzyme digest - (Jan/27/2009 )
scolix on Feb 4 2009, 08:32 PM said:
I would leave it for an hour. It should be plenty of time.
You could try site directed mutagenesis instead of error prone PCR.
You could try site directed mutagenesis instead of error prone PCR.
Hello,
Thanks for your reply. Would it be better to do a double digestion at the same time, or is it usually better to do a sequential digestion with PCR cleanup in between?
-jiajia1987-
rkay447 on Feb 4 2009, 10:19 PM said:
I did something similar in that I random mutagenized the gene by doing a gene specific pcr and then ligating into the vector. You would need to use primers that are specific to the vector your library is currently in since there are thousands of different open reading frames. Not sure why you would do this since libraries are used to isolate single genes or screen for specific genes. You then mutate the single cDNA you isolated in whatever experiment or screen you preformed (what I did). But, it can be done. Do the pcr, dpn1 digest the original template library, digest with your restriction enzymes, pcr purify or precipitate (note that you will products of many different sizes so gel purification won't work), ligate. Problem is you will most likely loose a number of cDNAs. Why not attempt "round the world" pcr where you amplify the entire vector using taq? This way each cDNA gets amplified and hopefully mutated. You just treat the pcr with dpn1, transform and amplify the mutated library.
We did this to try to identify binding mutants. We had no clue where to place a site-directed mutagenesis so we went on a fishing-expedition and just randomly mutated the gene. Did two hybrid screens and identifed clones that interacted the way we wanted. Isolated the cDNA and sent for sequencing to identify where the mutation occured. Mine had two mutations which we then tested individually by doing site-directed mutations. Managed to identify a single residue critical for interaction and function in the protein of interest.
We did this to try to identify binding mutants. We had no clue where to place a site-directed mutagenesis so we went on a fishing-expedition and just randomly mutated the gene. Did two hybrid screens and identifed clones that interacted the way we wanted. Isolated the cDNA and sent for sequencing to identify where the mutation occured. Mine had two mutations which we then tested individually by doing site-directed mutations. Managed to identify a single residue critical for interaction and function in the protein of interest.
Hello rkay447,
Thank you for the reply.
The main reason why I am doing an error-prone PCR, Dpn1 digestion, followed by a reamplification is because I am producing a mutated library for selection purposes.
I am provided with a fusion gene, which is linked to pET22b and I am doing an error-prone PCR to get mutated versions of that fusion gene. Those are the ones which I will be using for a selection, which is why I am not doing a site-directed mutagenesis.
And also, you mentioned "note that you will products of many different sizes so gel purification won't work". What does this mean? Plus, is PCR cleanup or gel purification better? From what I know, PCR cleanup cleans the small pieces of wanted DNA, but it does not necessarily give you the digested PCR products that you want to ligate to your digested vector (which can be achieved by gel purification). Plus, if you get other bands in addition to the desired band that you want, won't PCR cleanup still give you your digested PCR products together with products present in other bands?
I hope I am not confusing you here.Do advise me. Thanks in advance!!
-jiajia1987-
Ok, I think there is a little confusion here because of the term library being used incorrectly. A library refers to a mixture of cDNAs representing every (or most) of the genes being expressed at a single moment in time in an organism, tissue, cell line, ect. You take a sample of whatever you want to make the library from (cell lines, xenopus embryos, tissue sample, ect.) and run a reverse transcriptase reaction where every single mRNA present in the sample is reverse transcribed to DNA. This is then ligated into a vector to give the mixture of cDNAs. There are thousands of genes in the one sample which are all different sizes. This is why I was thinking you would get products of all different sizes. If you do a vector specific primer pcr of a library, you are going to amplify all of the thousands of genes which all have different sizes and therefore can not be purified by a gel.
However, it sounds like you are working with one gene and are trying to mutate the one isolated cDNA. Yes? Then I highly recommend you gel purify the pcr to eliminate the template plasmid, digest and pcr purify to remove enzymes and buffer. Quantify and ligate.
-rkay447-
jiajia1987 on Feb 4 2009, 04:29 PM said:
scolix on Feb 4 2009, 08:32 PM said:
I would leave it for an hour. It should be plenty of time.
You could try site directed mutagenesis instead of error prone PCR.
You could try site directed mutagenesis instead of error prone PCR.
Hello,
Thanks for your reply. Would it be better to do a double digestion at the same time, or is it usually better to do a sequential digestion with PCR cleanup in between?
If you are using compatible buffers, then I would do a double digest. Saves time.
-scolix-
rkay447 on Feb 5 2009, 02:08 AM said:
Ok, I think there is a little confusion here because of the term library being used incorrectly. A library refers to a mixture of cDNAs representing every (or most) of the genes being expressed at a single moment in time in an organism, tissue, cell line, ect. You take a sample of whatever you want to make the library from (cell lines, xenopus embryos, tissue sample, ect.) and run a reverse transcriptase reaction where every single mRNA present in the sample is reverse transcribed to DNA. This is then ligated into a vector to give the mixture of cDNAs. There are thousands of genes in the one sample which are all different sizes. This is why I was thinking you would get products of all different sizes. If you do a vector specific primer pcr of a library, you are going to amplify all of the thousands of genes which all have different sizes and therefore can not be purified by a gel.
However, it sounds like you are working with one gene and are trying to mutate the one isolated cDNA. Yes? Then I highly recommend you gel purify the pcr to eliminate the template plasmid, digest and pcr purify to remove enzymes and buffer. Quantify and ligate.
However, it sounds like you are working with one gene and are trying to mutate the one isolated cDNA. Yes? Then I highly recommend you gel purify the pcr to eliminate the template plasmid, digest and pcr purify to remove enzymes and buffer. Quantify and ligate.
Hi there,
I am sorry about the confusion. Yes, I am working with one gene, which is a fusion gene, and I am trying to mutate it so I can get a lot of copies of variants.
What I did was to digest my PCR products and gel purify the band which I wanted. Would it have any difference from gel purifying the PCR products first before digestion and doing a PCR cleanup? My supervisor told me that the advantage of a gel purification after digestion is that you definitely get the product that you wanted.
I did my ligation and did a PCR on the ligated product to see if ligation was successful. Ran a gel, but there were smears and a lot of unspecific bands, included the band for self-ligated vector. My gene library band is there, but I have no idea why there are so many other bands along with it.
Now, what I am trying to do is to rescue the library. I have done a transformation with the ligated products and will be doing a miniprep to prepare it for sequencing to see if I get the inserts in my vector and whether the vectors are mutated. -jiajia1987-
scolix on Feb 6 2009, 12:50 AM said:
jiajia1987 on Feb 4 2009, 04:29 PM said:
scolix on Feb 4 2009, 08:32 PM said:
I would leave it for an hour. It should be plenty of time.
You could try site directed mutagenesis instead of error prone PCR.
You could try site directed mutagenesis instead of error prone PCR.
Hello,
Thanks for your reply. Would it be better to do a double digestion at the same time, or is it usually better to do a sequential digestion with PCR cleanup in between?
If you are using compatible buffers, then I would do a double digest. Saves time.
I am using BamH1 and Nde1 on my PCR products while BamH1, Nde1 and Nco1 on my vector. They do not use the same buffers, but the buffer recommended for the combination would be NE Buffer 3 or BamH1 Buffer. Would you still do a double digestion?
-jiajia1987-
yes but u'l loose both sites in the process!!! (this is in response to the original question)
-arush-
arush on Feb 6 2009, 11:22 PM said:
yes but u'l loose both sites in the process!!! (this is in response to the original question)
Hi Arush,
which original question do you mean? Are you referring to the below?
"Hi everyone, I just wanna ask how long do you guys usually leave your enzyme digestion? Is 3 hours enough?
Has anyone tried making mutated libraries using error-prone PCR and doing an enzyme digestion on the PCR products so they can be cloned into vectors?
Thanks in advance. "
If yes, i did think that error-prone PCR will cause some mutations in the restriction sites. However, the mutation rate will not be so high that the restriction sites will be lost on every single copy. The mutation is also random. So, I will still get back some copies that are mutated somewhere else other than the restriction sites. Right?
-jiajia1987-
jiajia1987 on Feb 6 2009, 03:04 PM said:
I am using BamH1 and Nde1 on my PCR products while BamH1, Nde1 and Nco1 on my vector. They do not use the same buffers, but the buffer recommended for the combination would be NE Buffer 3 or BamH1 Buffer. Would you still do a double digestion?
According to NEB buffers,
BamHI -
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 100%
NdeI
NEBuffer 2: 100%
NEBuffer 3: 75%
NEBuffer 4: 100%
NcoI
NEBuffer 1: 100%
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 100%
As you can see, one could do a combined digest with either buffer 2 or 4. I might even do with buffer 3 as the enzyme would work 75%.
I agree BamHI comes with a special buffer but it definitely works with the other buffers equally well.
-scolix-
scolix on Feb 7 2009, 03:54 AM said:
jiajia1987 on Feb 6 2009, 03:04 PM said:
I am using BamH1 and Nde1 on my PCR products while BamH1, Nde1 and Nco1 on my vector. They do not use the same buffers, but the buffer recommended for the combination would be NE Buffer 3 or BamH1 Buffer. Would you still do a double digestion?
According to NEB buffers,
BamHI -
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 100%
NdeI
NEBuffer 2: 100%
NEBuffer 3: 75%
NEBuffer 4: 100%
NcoI
NEBuffer 1: 100%
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 100%
As you can see, one could do a combined digest with either buffer 2 or 4. I might even do with buffer 3 as the enzyme would work 75%.
I agree BamHI comes with a special buffer but it definitely works with the other buffers equally well.
I am not too sure why BamH1 comes with a special buffer. Is it because of its STAR activity? Maybe that is why I get my ligation but when I run a gel to check my inserts, there are many bands? But then again, when I do a colony PCR on the cells transformed with the ligated products, I get only one band, which is the insert band of the right size. I am puzzled at why these two gives different results.
And, I checked out NEB's double digest finder, it says NEBuffer 3 is the best buffer for the three combination, though I did think of using NEBuffer 2 or 4. But my supervisor told me to use BamH1 buffer, so I don't have much of a choice.
-jiajia1987-