Restriction site clonning failed - (Oct/05/2006 )
for the last month I am trying clone my 4 inserts into pET and pMAL vectors
I have incorporated res sites into my primers (huge mistake I have used taq pol for my PCR)
anyway I have obtained my 8 inserts I did a sequentional digestions with EcoRI/HindIII, XbaI/SalI, XbaI/XhoI with purifications in between for all my cloning products, visualized nbands are ok seems everything is fine so I do the ligation (rapid lig mix Roche) vectors 5.4kb 6.6 kb inserts 1.1 kb 1.6kb 3:1 (i/v) ratio and... just a few transformants both in neg controls and plates of interests (I did one trick even ( with digestion of ligatin mixtures 1uL with inserts noncutters enzymes which have restriction sites in the afterligation missin part of the vestors)
nothing!
One month!! its way too long! My boss told me she has 100% efficiency in one week!!
MY self esteem droped below marine level..
HELP PLEASE DEAR FRIENDS!
When you designed your primers, did you add the correct number of bases after the restriction site for optimal cutting by the enzyme?
well, this is trouble indeed.
Since cloning has many many possible places where things could go wrong, could you give the details of your cloning.
The primer design, the number of extra base pairs at the end, to allow the RE to cut. The approximate amount of DNA digested, the time digested, buffers used, enzyme quantities used, vokume of the digestion mix.
Any check gels conducted. (especially for the vector)
Cleaning up strategy for PCR product... gel extraction? Do you use kits? Or LiCl or agarase?
Ligation time and temperature and volume. Drying method. The condition of the ligase and ligase buffer. Can you confirm that the two are in good condition and have successfully ligated a vector in recent weeks.
Transformation method? Electroporation or chemical transformation.
And what do you mean by nothing? No colonies? Or no clones with the desired plasmid. Ampicilin concentration used.
Also please clarify the number of inserts you have? Is it 4 or 8? How is it for you to make more PCR insert. Taq is a very very bad idea. How many cycles did you run the Taq PCR reaction for.
Finally have you confirmed that you have DNA in the ligation mix just prior to transformation (by running a portion of the ligation mix on a gel)
Yes I have added 6 pairs to each of designed primers
Thank you for your answer! really
The primer design, the number of extra base pairs at the end, to allow the RE to cut. The approximate amount of DNA digested, the time digested, buffers used, enzyme quantities used, vokume of the digestion mix.
-6bp added to each 5' of primer
-sequentially overnight within the buffer appropriate for each enzyme (no star act observed no supercoiled band seen)
-50uL dig mix 40uL odf DNA (conc app 5 ug total, 50 units of ach enzyme)
Any check gels conducted. (especially for the vector)
-gels ok seems that vec is cut (sequential digestion products checked i.e EcoRI, HindIII, Eco/HindIII)
Cleaning up strategy for PCR product... gel extraction? Do you use kits? Or LiCl or agarase?
qiagen qiaqiuck gel purif kit each ligation substrate was purified
Ligation time and temperature and volume. Drying method. The condition of the ligase and ligase buffer. Can you confirm that the two are in good condition and have successfully ligated a vector in recent weeks.
Transformation method? Electroporation or chemical transformation.
chemical transformation
And what do you mean by nothing? No colonies? Or no clones with the desired plasmid. Ampicilin concentration used.
-no clones with desired plasmids (checked by PCR and double digestion)
Also please clarify the number of inserts you have? Is it 4 or 8? How is it for you to make more PCR insert. Taq is a very very bad idea. How many cycles did you run the Taq PCR reaction for.
-I have four inserts for pET and four for pMAL. With taq it was originlly my idea since I am beginner in cloning and when I have found out that taq is very bad idea all inserts were already made.
-30 cycles
Finally have you confirmed that you have DNA in the ligation mix just prior to transformation (by running a portion of the ligation mix on a gel)
-i have run the gel - nothing can be seen (20 uL total of lig mix - 4 ul withdrawn total Vec DNA 40ng Insert 40 ng - calculated acc to the common equation and assess from the gel run prior to ligation))
[/quote]
I think you need to make your inserts again using HiFI taq, if you do get the ligation to work and your inserts have a mutation you will probably have to start all over. I use only 25 cycles as well to decrease the possiblity of incorrect sequences.
For your restriction digests, 1-2 hr should be enough, no need to digest overnight.
One question.. after making your inserts, did you purify them before doing the restriction digest?
For your restriction digests, 1-2 hr should be enough, no need to digest overnight.
One question.. after making your inserts, did you purify them before doing the restriction digest?
Thank you very much for your answer!!
Yes I have purified them after each of sequential digestion (pirs just purify second gel-purify)
So do you think that there may be mutations in my restrition sites? but for all inserts? Maybe it sounds stupid but is it possible?
I am so so discourage and frustrated and sad!
Thank you for all your suggestions!
Yes it is possible there could be mutations in the restiction sites, but not likely for all of them. The problem with the restriction digests for the inserts is its impossible to tell if it worked. But I was wondering if you purified them not after the restriction digests but the PCR product. Did you use it directly for restriction digest or did you purify it first? The buffer from the PCR reaction could interfere with the activity of the restriction enzyme.
Yes I have purified from this enzymatic reaction as well.
I really do not know. Maybe problem is my hands. Nothing is working
and it seemed to be so easy at the beginning..
warm regards anyway and thank you.
Vector prep: combine 25 µg of vector (for instance, 25 mg of 2666 bp pUC9 equals 14.4 pmol or 8.8 e 12 molecules) with 20 µL of 10x KGB, 80 units @ of BamH I and Hind III, 3 units of Shrimp Alkaline Phosphatase and Type I water to a final volume of 200 µL. Incubate at 37°C for 3.25 hours. Small scale 20 µL reactions using single restriction enzymes should be performed and analyzed with uncut plasmid DNA and 5 µL of the vector digest on an analytical 0.8% agarose gel to confirm that both restriction enzymes are performing as expected. Overdigestion will allow contaminating exonucleases in the restriction enzymes to nibble the ends of the DNA fragments and prevent ligation. There is another caveat: determination of DNA concentration via UV/vis spectrophotometers can result in as much as a 10-fold error in actual DNA concentration. Use of a fluorimeter is much more accurate.
Insert prep: combine 14.4 pmol of insert (PCR product or plasmid-derived) with 20 µL of 10x KGB, 80 units @ of BamH I and Hind III and Type I water to a final volume of 200 µL. Incubate at 37°C for 3.25 hours. Small scale 20 mL reactions using single restriction enzymes should be performed and analyzed with uncut insert and 5 µL of the insert digest on an analytical native 12% acrylamide gel with appropriate markers to prove that both restriction enzymes are performing as expected.
Rather than thinking about restriction digest reactions in terms of micrograms of material, it is more useful to consider the pmoles of DNA, which relates directly to the number of molecules. If 25 ug of pUC9 equals 8.8 e 12 molecules, then there are 8.8 e 12 unique restriction sites that must be cut. But 1 ug of a 100 bp PCR product contains 9.1 e 12 molecules and a corresponding 9.1 e 12 restriction sites. 80 Units of restriction enzyme will cut 25 ug of pUC9 or 1 ug of a 100 bp PCR fragment in KGB in 3.25 hours at 37°C.
Similarly, it is much more useful to design ligation reactions based on the number of molecules rather than the number of micrograms. All of the 3:1 or 10:1 insert:vector ratios are based on using micrograms. What you should actually be concerned about is having an equal number of molecules of insert and vector in the ligation reaction. If you combine 30 fmoles of a 100 bp PCR product with 30 fmoles of pUC9, the 1950 pg of insert and 52 pg of vector result in an optimal 37.5:1 ratio. But 30 fmoles of a 800 bp DNA fragment combined with 30 fmoles of pUC9 converts to 156 pg of insert and 52 pg of vector resulting in an optimal 3:1 ratio.