Digestion, Ligation or transformation problem? - (Feb/20/2007 )
[quote name='netnus' date='Nov 30 2004, 01:17 PM' post='9023']
Hi all,
I have just joined the forum and I believe this is what I have been partly missing for success. I have been trying to do some cloning work for a while and life seem desolate! I am trying to get a 1440bp fragement into a 4007bp vector. I am using kpnI and BamHI enzymes from invirogen digested in buffer 4. I have tried both the sequential and mixed digestions and seem to get the right sizes after digestion. I am using a PCR product for the insert and just added the BamHI/KpnI sites to my primer ends and included 2 bases for each at the beginning. The PCR product is perfect. I go ahead to do the ligation using the T4 ligase from invitrogen. I am not sure of the ligation mixes because I just run a small gel of the digested product and I estimate the volumes based on the band sizes. I then go ahead to do the transformation using the heat shock method, 1 minute at 42C. I get no colonies in most of the plates and in some cases, i get a batch of them, only to test when I have the vector size back. I dont do any control though! Supringly, I some times get the insert size and I wonder how that is possible and in some cases, I even get fragements with sizes that I cant explain! I have done a couple of these and even tried using single enzyme digestion with KpnI, with the insert being a PCR product, all in vain! I am so dissappointed! I have done cloning with non PCR product and I was able to get the insert in my vector. However, I have not registered any success with cloning a PCR product in a period of 6 months! I need some help please!
Thanks,
Tricent.
Check that your ligase is really working? by religating a single cut vector for instance. ligase buffer is also important. freeze thaw cycles kill the ATP in ligase buffer. so if the buffer is not fresh then consider adding ATP into your ligation reaction.
have you tried different ratios for your ligation? have you had any self-ligated vector containing colonies?
do you think both of your enzymes are working? one of them may not be cutting well so that you cannot insert your fragment into vector.
lastly, when I do chemical transformation, I shock the bacteria for 90 sec at 42C and then keep it on ice for 2 min.
that's all I can say for now, good luck!
Okay, I have a few things you can try. Do not despair though, cloning is a major source of frustration in research (it took me 4 months to get my first construct when I had no research experience and was just starting out, now it's not a problem). At any rate:
-KpnI requires BSA -- I always have used NEB buffers, where I have to add BSA separately. Is BSA already in buffer 4? If not, you may need to add it.
-I digest for at least 2 hours. During the last 20 minutes or so, I add 1-2uL CIAP (phosphatase) to the vector tube to dephosphorylate it. This helps minimize self ligation of the vector.
-More is not better. When estimating concentrations, I usually never use more than 5uL of each vector and insert for my ligation. Too much will not ligate efficiently. Similarly, too much DNA in your digests will not digest efficiently.
-Has someone else used your ligase and had it work? We had one "go bad" on us (our freezer went out for a few hours...), and had to get new. We use Epicentre Quick Ligation Kit. Do you need to add ATP to your T4 buffer or is it already inside?
-After digestion, how are you purifying? I run the entire digests on a 1% agarose gel and use Qiagen's gel extraction kit. This removes the enzymes, most importantly.
-Have you tested your competent cells to be sure they are accepting plasmid well and colonies appear on the same plates you are using (i.e. antibiotic is not too high)?
-Finally, I always do a vector only control. That is, I set up two ligations. One with vector, insert, buffer, ATP, ligase, and one with water in place of insert. I transform no more than 5uL of my ligation mixture into my cells (25min. on ice with DNA, 45sec. heat shock (less if I bought the competent cells), 5min. on ice, add 1mL LB and recover at 37 (unless it is a temperature sensitive vector) for 1 hour). Then, I gently spin down my tube, remove most of the sup, and resuspend the whole pellet gently into the small amount of media left (normally about 100uL). I plate the entire vector only and normal ligation. Since your plasmid should not reanneal if cut by both enzymes, you should get minimal colonies on your vector only (you will get some if the plasmid is only cut once), and more on the ligation plate. If this isn't the case, I don't even check my colonies.
I hope that gives you some things to consider...good luck!
I usually leave 5 bases on each primer to allow proper digestion. Many times 2 is sufficient but one can never be too sure.
Does your vector have inverted repeats which is complicating things for you?
Ok, some suggestions...
As scolix said, 2bp either side of a cut site ain't much. I don't really like cutting the ends off PCR products like this, as there's no way to tell if the digest has worked or not. Also, what cells are you using that need to be heat-shocked for 1 minute? Most commercial cells only need 30 secs, and efficiency drops right down after 40secs or so.
I think you need to try and work out where this whole process is going wrong. Firstly, try cloning the PCR product into a TA vector, then see if you can cut it out. That will tell you if the digest is working.
When you transform, include a control vector reaction like pUC18, which comes free with most kits. You should get heaps and heaps of colonies from the control reaction, if not then something is wrong with the transfromation process.
Ligations are a little harder to troubleshoot. Try re-ligating a linear vector as a control, you could even try a PCR on the ligation mix to check for the right construct, I have got this to work before.
Good luck...
have you tried different ratios for your ligation? have you had any self-ligated vector containing colonies?
do you think both of your enzymes are working? one of them may not be cutting well so that you cannot insert your fragment into vector.
lastly, when I do chemical transformation, I shock the bacteria for 90 sec at 42C and then keep it on ice for 2 min.
that's all I can say for now, good luck!
Thanks so much dodosko for the response! I think I will need to do a ligation test that way. I once run a gel with the ligated product but I saw seperate bands for the vector and insert and one time a very high band, higher than the vector and insert combined. I think the buffer works because I even tried using a new tube, opened the first time. Yeah, I also try making different ligation ratios but none gives me the disired product! In most cases, I get the vector fragement (self-ligated) after checking the product. I do believe my vector is well cut but I am not sure of the PCR product. I use two enzymes kpnI and BamHI and BamHI has two sites and I am able to see the 3 bands on the gel, so that way, I assumed the digestion worked. I have not tried heatshocking for as long as 90 sec. I have only been heatshocking for 1 minute at 42C, and also 3minutes at 37C.
Thanks for the suggestions, will try to adjust a few things.
Tricent
-KpnI requires BSA -- I always have used NEB buffers, where I have to add BSA separately. Is BSA already in buffer 4? If not, you may need to add it.
-I digest for at least 2 hours. During the last 20 minutes or so, I add 1-2uL CIAP (phosphatase) to the vector tube to dephosphorylate it. This helps minimize self ligation of the vector.
-More is not better. When estimating concentrations, I usually never use more than 5uL of each vector and insert for my ligation. Too much will not ligate efficiently. Similarly, too much DNA in your digests will not digest efficiently.
-Has someone else used your ligase and had it work? We had one "go bad" on us (our freezer went out for a few hours...), and had to get new. We use Epicentre Quick Ligation Kit. Do you need to add ATP to your T4 buffer or is it already inside?
-After digestion, how are you purifying? I run the entire digests on a 1% agarose gel and use Qiagen's gel extraction kit. This removes the enzymes, most importantly.
-Have you tested your competent cells to be sure they are accepting plasmid well and colonies appear on the same plates you are using (i.e. antibiotic is not too high)?
-Finally, I always do a vector only control. That is, I set up two ligations. One with vector, insert, buffer, ATP, ligase, and one with water in place of insert. I transform no more than 5uL of my ligation mixture into my cells (25min. on ice with DNA, 45sec. heat shock (less if I bought the competent cells), 5min. on ice, add 1mL LB and recover at 37 (unless it is a temperature sensitive vector) for 1 hour). Then, I gently spin down my tube, remove most of the sup, and resuspend the whole pellet gently into the small amount of media left (normally about 100uL). I plate the entire vector only and normal ligation. Since your plasmid should not reanneal if cut by both enzymes, you should get minimal colonies on your vector only (you will get some if the plasmid is only cut once), and more on the ligation plate. If this isn't the case, I don't even check my colonies.
I hope that gives you some things to consider...good luck!
Thanks Cheamps for the response!
I think the vector is well digested because I do get the 3 bands that I expect and with the correct sizes, but I am not sure of the PCR product! I also use the Qiagen gel extraction kit with beads and use a 1% or some time 0.8% gel. However, I have not been using the CIAP at all and maybe this is something I will have to consider in the next steps. In the past, I think I was using alot of DNA because I was basing on the gel results to do the ligation. However, in the last ligation, I changed to using the molar ratios 3-90 fmol, setting up 3 reactions: 3V-9I, 15V:45I and 30V: 90I but all in vain! I also changed the ligase recently, so it should be fine, but I dont know whether the reaction takes place. My transformation is 15-30 minutes ice, 1minute heat shock at 42C and 1 minute ice and then add 1ml Lb and shake at 37C for 1hour. I dont know whether that's a good one! I use ampicillin at 100mg/L on plates. In most cases, i dont get any colonies and then at times, i get a few, vector size and even insert size! One time I did a PCR with the product, I got the insert fragement on the gel but when I digested with the enzymes, there was nothing cut and I wondered what that was! By the way, is it possible to get insert-containing colonies and if yes, how is that possible and yet it doesnt have the resistance gene? Then finally, i plan on taking on the Topo TA cloning using the invitrogen but I dont know how it will work! I think it's a little easier.
Some good things I need to take into consideration.
thanks so much!
Tricent
Does your vector have inverted repeats which is complicating things for you?
Thanks for the response!
I added the 2 bases on each using the invitrogen catalog's recommendations. One of my sets I actually have 6 bases and this one I am using one enzyme (EcoRI) but it also failed! By the way, how do I know that my vector has inverted repeats? Sorry, i am just new in this field.
Thanks,
Tricent
As scolix said, 2bp either side of a cut site ain't much. I don't really like cutting the ends off PCR products like this, as there's no way to tell if the digest has worked or not. Also, what cells are you using that need to be heat-shocked for 1 minute? Most commercial cells only need 30 secs, and efficiency drops right down after 40secs or so.
I think you need to try and work out where this whole process is going wrong. Firstly, try cloning the PCR product into a TA vector, then see if you can cut it out. That will tell you if the digest is working.
When you transform, include a control vector reaction like pUC18, which comes free with most kits. You should get heaps and heaps of colonies from the control reaction, if not then something is wrong with the transfromation process.
Ligations are a little harder to troubleshoot. Try re-ligating a linear vector as a control, you could even try a PCR on the ligation mix to check for the right construct, I have got this to work before.
Good luck...
Thanks Scrat for the response!
Yeah, i only added the two bases because I used the gudelines in the invitrogen catalog for that, but even in cases where I added 6, I still had problems and I have to give it a break. i use my own chemical made cells, but I have been testing them only on a known plasmid and I have been able to get lots of colonies apart from a few times, then I discard and make others. I dont know whether this works best in testing! I am also looking at the possibility of using the Topo TA from invitrogen and see if i can get a change. I am not sure where the problem is because I am able to clone non-PCR fragements, just those that are digested. i will try first the ligation test and then transformation because, I feel this is where the problem lies. But I cant tel what's wrong with the ligation because the ligase works, other people have used it recently but maybe my reaction has a problem! How do you mix your ligation reaction? Do you use gel estimations or molar ratios and which ones?
Thanks and regards,
Tricent