I have a gene(wild type) cloned in pBAD18 Kanamycin resistant plasmid. The gene is 2185 bp. And the plasmid is 5437 bp. I wanted to insert a 6X His tag at the C-terminal end of the protein of this gene. I found that the last codon codes itself for a His. So I followed the general method for designing a primer i.e.: Last codon(CAT)-6X His codon (CAT or CAC)- Stop codon-XbaI recognition sequence. The primer length is 30 bp with a Tm of 62 degrees approx and the forward primer I used was 28 bp and has a EcoRI recognition site with Tm 59 degrees approx.
I have tried amplifying my gene using a gradient PCR vaying the annealing temperature (see gel image). I expect to get the product in between 2Kb and 3Kb(2203 bp). But I have a range of variation in amplification above 10 Kb. What is getting amplified is my question? Is my gene at all getting amplified? I dont think so. What is going wrong.
I am using Pfu polymerase with 30 steps amplification. I have done gradient PCR with variation in annealing please see gel image attached. PCR program:
Initial denaturation: 95 degree- 3 min
Loop:30X
denaturation: 95 degree- 30 sec
annealing: gradient- 55- 64 degree- 30 sec
extension: 72 degree- 3 min 30 sec
Final extension: 8 min
Store: 4 degree.
Notice the amplified product that I am getting is above 10 Kb.- DNA Ladder used is 1Kb NEB ladder.
please suggest ways I can get my gene amplified for cloning. I want my insert to have a 5'-EcoRI and 3'-6X His-XbaI.
I think you should try this PCR programe once :
Innitial denaturation : 94C in 2m
10 cycles:
denaturation: 95 degree- 20 sec
annealing: 50 degree- 30 sec
extension: 72 degree- 2m
20 cylces:
denaturation: 95 degree- 20 sec
annealing: 55 to 60 degree- 30 sec
extension: 72 degree- 10m
Final:
72 unlimite!
It may work!
Thanks Sezer. I have not quantified my plasmid as of now. But I am using 1ul of the plasmid. I will use 0.5 then and anneal with 57 degrees and 35 cycles as you suggested. But tell me is there any wrong in the primer design?
But, if you see my primer design the pBAD18 plasmid has a XbaI and my primer actually binds to the 3 bases there and the XbaI site, with the 6X His tag bases in the middle. Can this be a fault?
That's way too much plasmid. You should dilute a standard miniprep at least 1:100 for PCR, and then use 1 ul of that dilution.
No, your gene was not amplified. Have you checked your gene had successfully cloned into the plasmid by restriction digestion? And look like you may have some genomic DNA contamination too.
Hi Amlan,
The strategy you have planned works well generally, however as mentioned above ou should take care of the following things
1) The primers length, especially the region perfectly complementary to the end of the gene should be sufficiently long (15-20 nt). The extra regions added (such as His tag and restriction sites) should be considered as they will not bind to the plasmid.
2) The upper bands looks plasmid (added as template) you are adding too much. This much is not required for PCR.....the culprit may be the primer designing
3) You may linearize the plasmid before adding it to the PCR...use restirction enzyme at a region away from the gene..
it should work
There is some 2k amplification already at 57 and 58 degrees. You can cult gel to recycle it.
one thing I noticed is the way you calculate the primer TM, only the recognition sequence should account for it. As in the starting cycles, only these sequence will anneal. Another thing is that your primer is too long, which will reduce amplification efficiency a lot.
And I agree with above comments that your plasmid DNA is too much, giving you that 10k bands.
Thank You Van for your suggested PCR program I will give it a try.
Thanks a lot Fang. As to the primer it has to be that much long. You see, there is 6X His that counts for 18bp, the last codon of the gene: 3bp, a stop codon 3bp, an XbaI seq. of 6 bp and that totals to 30bp. How will I reduce it?
As suggested here by Ajay Saini, if I use 15-20 bp of the gene as the primer and then use the 6X His and XbaI recog. seq. that will make at least (15+18+3+6)= 42bp primer. Its going to be too large. So will that be feasible? Fang you say that my primer is already too large. Then what can be done.
Hi Sezer,
In my lab I do have a pET22b vector. It has a His tag in itself. My gene (wild type) has a NcoI and SmaI site at the 5' and 3' ends which I had checked by digestion. It did yield me a band at the 2 Kb location. I have the the digested product. To add on, I had use two sequences as primers before and they had given me an amplicon of 2 Kb i.e. the exact gene. I had eluted that band out from the gel too and have the PCR product as well.
Will you please suggest me strategies hence to clone in pET22b?
As far as I understand, your current primer has 3 bp for CAT and 3 bp for Xbal, then in the middle you also have a 6x His sequence that you want to insert, is it the case?
If so, the problem is not the length of primer or concentration of DNA. As others suggested, you should have a gene targeting primer which can recognize at least 15-20 bps with a overhang of 18+3+6.
The former design has only 6bp binding to template and has a 6x his sequence as a hairpin in the middle, which basically won't bind to your template at 55 degree. The calculation of annealing temperature doesn't need those overhangs or hairpin structure. So in this case, even a (15+18+3+6)= 42bp primer, only the 15 bp accounts when used for calculating annealing temp.
Dear Amlan,
I would like to suggests the following things.
1. As suggested by Fang, recheck the primer length and calculate the Tm
2. Use Touch-Down PCR instead gradient to check whether the gene is getting amplified with appropriate expected size
3. Please use any high-fidelity Taq Polymerase (in my case, I use Sapphire PCR master mix from TaKaRa), then later on you can go ahead with Pfu.
4. If you want to continue with Pfu enzyme, try increasing the extension time from 30s to 1-2 min because you are intent to amplify larger target
5. What is the percentage of your agarose gel?
6. Are you sure that you are using 1kB ladder, if you are using 100bp ladder, then the PCR amplicon will be above the last marker band. Check the ladder once again
All the best
Rajkumar
Dear Amlan,
1) how much of your starting plasmid template do you use for PCR? Have you tried to load it on the agarose gel to check that the bands you observe are not the original template?
2) it seems to me that 30 nt are too short for your primer: it must contain 6 nt for the restriction site, 3 for the stop codon, 5(or 6) x3 His codons = 15-18 and finally the sequence complementary to your target... try to extend the complementary part at least to 18-20 nt.
(and beware you must not indicate a primer in bp, as it is single stranded! ;-))
good luck!
Paola
- Badges
- Science method