I am trying to amplify the luminal domain of TRAP alpha subunit (ER), but probably TM is too high. I performed PCR with two different pairs of primers but I get bands below 500 bp (see picture), and also sequencing does not match completely. The domain of alpha that I want to clone is 560 bp. Can somebody help me? A.Russo
First pair of primers
foward: 34 nucl. 5' extension, Ned I site
reverse: 29 nucl., 5' extension, Sal I site
Second pair of primers
Fow: 47 nucl., 5' extension SgfI
Rev: 30 nucl. 5' extension XhoI
Polymerases: Pfu and Phusion High Fidelity
Template: pCMV6-entry Vector (4.9 kb) with the entire cDNA TRAP alpha (864bp)
HI Antonietta,
I gather from your first post that you're incorporating a restriction site into the PCR product using overhanging primers? When calculating your Tm, make sure you only account for the region of the primer which is actually binding to the template, rather than using the Tm based on the full length of the primer sequence. It is likely that the full sequence will have a higher Tm than just the template-binding region. Therefore, if you use this higher Tm for annealing, it's likely that you will get inefficient binding which could then lead to the introduction of errors in your sequencing.
I hope that makes sense. I used to do this sort of PCR cloning a lot and always calculated Tm based on just the template-binding region of the primer and never got any errors in my sequencing.
Thanks,
Mark
What is your DNA ladder and what were the parameters you used for PCR (ie. denaturing temp and time?, annealing temp and time, and extension time?).
1kb Gene Ruler Ladder (see file)
Pfu Polymerase
95 °C 30 sec
95 °C 30 sec (35x)
53 °C 30 sec (35x)
72 °C 45 sec (35x)
72 °C 2 min
Volume reaction 100 µL (picture that already loaded): first pair of primers.
95 °C 30 sec
95 °C 30 sec (35x)
53 °C 30 sec (35x)
72 °C 55 sec (35x)
72 °C 2 min
Volume reaction 100 µL (picture 2): first pair of primers.
Band on the gel is right but sequencing did not match completely.
Phusion HF Polymerase
98 °C 30 sec
98 °C 10 sec (35x)
72 °C 30 sec (35x)
72 °C 20 sec (35x)
72 °C 5 min
Volume reaction 20 µL (picture 3): second pairs of primers
98 °C 50 sec
98 °C 10 sec (35x)
72 °C 30 sec (35x)
72 °C 20 sec (35x)
72 °C 5 min
Volume reaction 20 µL, + DMSO and CG buffer (similar to picture 3)
98 °C 3 min
98 °C 10 sec (35x)
64.7 °C 30 sec (35x)
72 °C 17 sec (35x)
72 °C 5 min
Volume reaction 20 µL (similar to picture 3)
Thanks
HI Antonietta,
I gather from your first post that you're incorporating a restriction site into the PCR product using overhanging primers? When calculating your Tm, make sure you only account for the region of the primer which is actually binding to the template, rather than using the Tm based on the full length of the primer sequence. It is likely that the full sequence will have a higher Tm than just the template-binding region. Therefore, if you use this higher Tm for annealing, it's likely that you will get inefficient binding which could then lead to the introduction of errors in your sequencing.
I hope that makes sense. I used to do this sort of PCR cloning a lot and always calculated Tm based on just the template-binding region of the primer and never got any errors in my sequencing.
Thanks,
Mark
I would expect these primers that anneal at a high temperature to anneal at a lowered temperature as well and lowering the temperature will not get rid of the wrong band that you have. What you may have is first cycle copy error. Doubly unfortunate that the product is also shorter because you can't limit this off target amplification buy cutting down elongation time. I would try these; see if you can add specificity to the primer by adding to the 5prime side. As Mark has pointed out you probably can add on and be in this range for Tm. Alternatively you may need to amplify replicate samples over a range of Tm and run a long gel to analyze it. Set up a few reactions and have Tms lower than what you are using. I suspect that at some lower temperature you will get both bands. You could then gel purify the correct mass band and use it for template. If you decide to do this you can wrap it up with a nested PCR type reaction. Amplify the 560 purified band with the RE site primers to obtain the quantity of product you need to cut and clone with the RE linker primers.
Hallo many thanks for your answers
In the last days I followed the piece of advice of Mark and also I reduced the extension time from 20 sec to 10 sec, template (1 pg in 20 µl reaction) and polymerase.
I got the right band (see picture) now Il will perform cloning as I did with the other three subunits of TRAP complex (it worked very well): digestion, purification, ligation, transformation, plasmid purification and sequencing.
I will let know what the results........
Picture, first band: template 1 pg, second band 45 pg. Also the amount of template has big role.
More precise I did PCR purification, digestion (5' extension enzyme sites), gel purification, ligation......etc.
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