I used gene specific primer to amplified the gene which is almost 4000 kb in length but i got smear on gel after trying the Mgcl2 gradient and Tm gradient. I got smear with Mgcl2 conc= 3.5, 4,4.5 at 56 degree and also at different tem 54.3, 55.4, 56, 58 and 59 using the constant 3.5 mgcl2 concentration.
the PCR program with 30 cycle
94 degree=3 min
94 degree=30 sec
tm (as mentioed above)
72 degree = 2.3 min
72 degree = 7 min
4 degree = hold
Dear Hari,
check the quality of template DNA and purify or dilute (5 - 10 times) if needed
Initial Denaturation step - 2 mins (qiagen link)
Denaturation step - few seconds
Extension 1 min/kb
MgCl2 1.5 mM will be sufficient
Annealing temp gradient ± Tm
The following links may help...
http://www.qiagen.com/resources/molecular-biology-methods/pcr/#Amplification of long PCR products
https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530
http://www.protocol-online.org/biology-forums/posts/7436.html
All the best.
What is your predicted Tm for the primers? You may find it helpful to increase your gradient up to 70 degrees (rather than max of 59). It is also good to have a high quality Taq for a amplicon of that length, it may not always be necessary to use a taq with proof-reading capabilities, depending on what you are using the fragment for, but it would be good to know what you are trying to amplify it with. The extension time will also depend on the taq polymerase you use - check the product manual for recommendations on that. Good luck!
It would be more useful if you could add a photo of a gel, but with that info, I can recommend you:
1. To diminish the MgCl2 to 2.5mM, the more concentrated the MgCl you use, the more probabilities that you can have smears due to an "overworking" Taq Pol.
2. Specially for this case that your product is about 4kb, your are trying to do a Long-PCR, so you Taq Pol should have some additional features if you want a good clear product (High-fidelity and proofreading activity would be great as mentioned above, depending on the purpose of the next assays too).
3. Also, the elongation time must be proportional to 1kb / 1min, so you need at least 5mins to assure the complete elongation.
4. The dNTP concentration should be considered to obtain a good product (>250uM dNTP's) https://www.neb.com/protocols/2012/10/15/m0323-longamp-taq-dna-polymerase-protocol This protocol have 300mM final concentration of dNTP, but I think that you can optimize on your own way.
https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase (Higher dNTP concentration)
I would first say a photo would be useful, as there can be many types of smears. In my experience of a smeared product I found that reducing the primer concentration helped. Also, how was the template purified? Is there a possibility of protein contamination from the extraction? In this case, adding 10x BSA (1ul per 10ul rxn) may help reduce smearing.
You are using too much MgCl2! 1,5 mM - 2.0 mM could be sufficient. Moreover you can usa a kit for Long Amplification (es. Expand Long Template System, Roche).
Thank You very much for your good suggestions, i am going to upload the pic of gel which i found
1) pic 1 on 13 augst is of gradient of DMSO and remaing component was constant
2) pic 2 on 14 augst is Mg cl2 gradient from (3.5, 4, 4.5, 5 and 5.5 ) earlier i used the mgcl2 conc. from 0.5 to 4 with 0.5 interval and got smear in wells with mgcl2 3.5 and 4 micro liter in 25 micro liter reaction volume
3) pic 3 on 16 showing the result of tm gradient (53.3, 56.2, 58.4, 59.4 and 59.9) with fix mgcl2 =3.5 micro liter
Hello,
I have experience of resolving this problem by touch-down PCR, I don't know that it is applicable to your case but it is said that touch-down PCR is applicable for up to 10 kbp amplification, smear band, etc.
here is the cycle:
1. 98 degree=2 min
2. 98 degree=10 sec
3. 74 degree (depending on primer tm)=60sec./ kb
4. 2-3 cycle for 5 times
5. 98 degree=10 sec
6. 72 degree (depending on primer tm)=60sec./ kb
7. 5-6 cycle for 5 times
8. 98 degree=10 sec
9. 70 degree (depending on primer tm)=60sec./ kb
10. 8-9 cycle for 5 times
11. 98 degree=10 sec
12. 68 degree (depending on primer tm)=60sec./ kb
13. 11-12 cycle for 15-30 times
14. 68 degree = 7 min
15. 4 degree = hold
denature 98 degree is for the template in case of being GC rich. the annealing+amplification temp is stepped down so that the primer specificity can be maximized. The Tm of the primer that I used was 65 degree and it worked well.
Adjust the cycle with your experiment and if you have further question, feel free to ask.
Good luck!
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC328507/pdf/nar00094-0209.pdf
Touch-down PCR is a good idea!
Anyway, are you sure your primers are well synthesized? It could be a bad synthesis.
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