Would u plz give me some suggestions? - (Jun/02/2006 )

Life is not always good, I think I am worried and depressed for a long time. It makes me really unhappy these days. Now, as a senior student, I have do some research work in one laboratory, which is called thesis for graduation.Now, I need to do is to constuct one gene vector. At first, I was very excited to do the experiment all by myself, I wrote a specific plan, enzyme cutting, electrophoresis, gel purification, ligation, transformation, and so on. Unfortunately, every time, after I do the transformation, and incubate the culture in the 37 degrees for 12-16 hours (the culture is Amp+), there is nothing on the culture, not a single recombinant! So then I did the experiment again at very beginning. And I did "thousands of" enzyme cutting, electrophoresis, gel purification, but there is never ever a a single recombinant after transformation!

I had discussed with the students in my labortatary, there are all very nice, and also I had used blunt-end or cohisive-end ligation, but still no recombinant. I know I should be patient and try to find the reasons, but everytime, it seems that new problem will come up, sometimes it is due to my careless or lack of knowledge, sometimes, it is the problem of the enzyme, plasmid and also the competent cells, so this experiment seems endless.

I think I should believe that I can make it, but as the time goes, I really do not have the confidence, what I do is doing enzyme cutting, electrophoresis, gel purification repeatedly, everyday, but no result, I think I will go crazy like this.
I do want to change this bad mood, but I do not have courage, but I now know, doing research is really difficult, I even wonder why I choose to do reseach, am I suitable?


Would you please give me suggestions, I bet next POST will not be so long.

Things can go wrong everywhere.

So, please tell us exactly what you are doing, what are your positive controls, how do you quantitate your DNA after extraction from gel, have you checked competence of your cells, what about your negative controls etc...

well, the thousand of cloning problems on this forum, and my particular experience of cloning shows that this exp can drive dizy . I've stucked for 18month and that's not finished.
For ligation, you can directly electroporate 1µl of your 10µl ligation.

You can use PEG during ligation, prepare new stock of competent cells....
I have tested sthg which gave me 4 clonings.
Instead of digestion/gel purif ligation, i just PCR my vector and the insert. Column purif, digest, column purif again (and you may check if your enzymes cut in primer extension mix at neb website) and ligate.
I think sthg goes wrong with the gel purification step.Can't figure out what it is really, but now i don't pass on gel, i have clonings that goes good.
The problem is now switched : you have to obtain PCR which is not that evident (i though it was a routine step, btu i have some problem with tough amplifications... )

Cloning tortures one and all. Had similar moments with cloning. U will overcome it and will make u mentally stronger.

Post step by step strategy of ur cloning, may be we can suggest some tips.

Thank you very much to varius, fred 33 and Scolix:)

Now here is my specific information of my work.
(1) Vector
It is about 10 kb, which can be obtain by double enzyme cutting from a 13 kb plasmid, here is the reaction volumn:
DNA 2 (the concentration is 0.8μg/μL)
MluI 1.5
EcoRI 1
Buffer 5
Add water to 50μL, 37 degree for 8-9 hours, and electrophoresis, I do see two fragments, but the 10kb fragment is not very clear, but bright, it seems so close to the 13 kb plasmid, and as we know, the plasmid is consisted of 3 parts, thus it is differcult to judge whether the 10kb pragment is what I want or the part of the plasmid, anyway, I just cut it and do the gel-purification.

(2)Interested DNA
It is 2kb, and Now, I use the PCR reaction to obtain it. Cause the vector has the MluI site, thus when designing the forward primers, we add the MluI site before it, however, there is one problem, the 2kb-interested fragment itself has two exactly same sequences, thus when we use the forward primers, it will definately bind these two parts, so we know, we will get two PCR product, one of which is wanted. The Tm is 70 by the software, we use the Software Dnassist2.0 and Oligo.

As the plasmid is 0.5μg/μL, I dilute it for 200 times before use. And I take 1μL from it as the template.
Template: 1-4μL(I tried 4 times, each time is not good)
Up: 1
Down:1
Buffer: 5
dNTP: 0.4 (25mM each)
Add water to 49 μL.
Add Pfu Enzyme 1 when the temp is 94 degree.

The Procedure is:
1.94 2min
2.94 30sec
3.58-62 30sec
4.72 2min
5 GOTO 2 30times
6.72 7min
7.4 forever

And after I did the PCR the first time, I really saw the 2kb fragment, but it was really very DIM, compared the other bright fragment. I was suggested to cut the 2kb fragment, and did the gel-purification, and used it as a template again, do the PCR reaction, also I increase the temp from 58 to 62, however, this time, nothing 2 kb fragment can be seen!! How can I do? Guess what, I do the 3-4 same PCR reaction simultaneously, and after electrophoresis, I cut these 3-4 fragments in one column during the Gel purificaiton, in order to increase the concentration, and it really DOES, however, I used over all the Pfu enzyme, which is very expensive, and be blamed and I was very guilty, what I should do is to find the optimal conditions, rather do many SAME PCR reaction! Now, as the pfu enzyme is used out,I can not continue the experiment, maybe I can borrow it from other labs, who knows.

(3) Ligation
Before ligation, I do the electrophoresis to see the ration of brightness of the vector and interested DNA. Due to the PCR reaction which is not ideal, the interested DNA is very dim, about 1/4 of the Vector. And I thought I was doing the blunt-end ligation, thus I thought the mol ration of Interested dna to vector should be 5:1or even 10:1, rather than 3:1. Thus I did a 5:1 and 10:1, ligation is

Interested DNA
Vector:
T4 Liganase 1
Buffer 1
Add water to 10μL, I almost add PEG8000 to it cause I thought it was blunt-end, luckily I stopped.

Then 12 degree for 8 hours, and transformation. it is Amp+, and I incubate it in 37 degrees for 12-16 hours, but none recombinant is showed.

(4) Competant Cells
Several mothes ago, our lab used over all the competant cells, and then they make some, they are very effective, however, we used over of them, and this time, my collegues made again, they use the PUC19 to test the effiency, they use 1pg to do the transformation, and only 20 recobinant, and the positive/negative control is ok, that is to say the effiency is not high, cause last time theirs is 100+ recombinant.

Well, these are my current work, now I am still worried about the Pfu enzyme, although we orded it, it will arrive in 1 months, and how to make sure the 10kb vector is not the part of the plasmid? How to pcr the bright 2kb interested fragment? How to ligate the right dna, and when can I see one single recombinant? Still do not know.

I think this post is longer than last, I bet next will be ok

Thank you all:)

I am worried abt ur digest with Mlu I. I had a bad experience with Mlu 1 in my old lab and my present lab also had similar problems. The key to Mlu 1 digestion is short digestion time else the ends are somehow chewed up by the enzyme and u cannot get the cloning going.

From ur digest protocol for the vector, i would suggest using only 0.5µl of Mlu1 and for only 40-60 min. EcorI might not b a problem.

Also u seem to have a PCR product so I guess try to digest them again shorter time points with MluI and try ligation.

I usually ligate using T4 ligase from NEB in RT for 30 min (for sticky end ligation). It might sound ridiculous, but it has worked for me. Try to get better cells if possible.

Hope this helps. There might be many other things which we can trouble shoot.

Good Luck !!!

Thank you very much, scolix

Now, the post-doc who guided me said, I should do some experiments to find the optimal condition for PCR reaction, cause the 2kb fragment is too dim

And also thanks for your suggestions on MluI, it is from NEB, and u said 40-60mins, but I did the double digestion, and meanwhile I use the EcoRI, but 40-60 seems too short? The unit of MluI is only 10U/μL, can it be possible:)?

Hi sunnylyon,
First of all, dont get bogged up much by ligation problems. Its just time and suddenly it clicks! You will get many clear colonies of +ve and no colonies on only vector. TIll that happens try these (might help):
1. Vector: Do restriction digestion for single enzymes along with double digestion to confirm that both MluI and EcoRI are cutting (Use the same buffer for both which you use in double digestion!)
You should see linearized bands in all but the size may very(13kb,13kb and 10kb)
2. For standardizing the PCR conditions do a series of experiments varying different criteria such as: dNTPs, annealing temp, pfu/Taq, # of cycles, duration of extension step(I use 72C for 1min-45 sec!!) , buffer, etc.
3. If you have problem with MluI (both as enzyme and in PCR amplification) then try doing ligation in which only one end blunt(MluI) and EcoRI is cohesive.
3. If you are NOT using quick ligase then I suggest to do ligation incubation at 16C for 24 hrs(works best for me)
4. and though the comp cells you have looks fine try using much more efficient ones.( I hope amp concetration and other transformation stuffs you've double checked!)
All the best!

I know I used the same MluI from NEB. I switched to invitrogen for MluI only, and it chnaged everything.
It might sound too less but as per the book, 1µg of DNA can be digested in 1hr with 1 unit enzyme.

u can give it a try with MluI, 0.5µl - 40-60 min.

As Calvin suggested try single digests to confirm if ur enzymes r working.

Good luck !!!

Thank you very much to Calvin* and nice Scolix again

Well, as Calvin* said, I may try to do the single enzyme digestion along with the double digestion, and you said "You should see linearized bands in all but the size may very(13kb,13kb and 10kb)".so what does these two"13 kb " mean, are they plasmid or linearized bands?

Scolix, although it is said in the book, "1µg of DNA can be digested in 1hr with 1 unit enzyme. ", I think maybe it can not work for all the enzymes, in our labortary, we never cut the enzyme in so short time, and in the NEB book, it is said in the NEB
Catalog, in Page 241"1.00, 0.50,0.25 and 0.13 units of restriction endonuclease wee added to 50μL reaction mixtures containing 1μg of unit-assay substrate DNA.....The minimum number of units that result in complete digestion of 1μg of substrate DNA in 16 hours was determined by agarose gel eletrophoresis."
And as for MluI , it is said the minimum number of units that resulted in complete digestion of 1μg of substrate DNA in 16 hours is 0.13, thus, when we shorted the time from 16 hours to 3-4 hours, so we can increase the units from 0.13 to 10, that is the traditional deed in our labortary, so Scolix, u mentioned the Invitrogen, does it the same company as NEB (I know little about that)?

Well, thank you again!~