PCR to amplify my insert not working - (Feb/08/2012 )
Im not used to doing PCR reactions so would appreciate your advice if you have any!
I am trying to change the tag on my plasmid from a Myc to a HA. In order to do this, In order to do this, I digested an empty vector containing the HA tag, and right now, I am trying to amplify my insert with the appropriate oligos.
My mentor has his own oligos for the N term and C term region and I also have a pair so I have been running both at the same time.
In order to run the PCR reaction, I am using a product called "Takara PrimeSTAR HS DNA Polymerase". The protocol they give is pretty straight forward in terms of how much to add in the master mix.
5X PrimeSTAR Buffer - 10 ul
dNTP - 4 ul (Final conc: 200 uM)
Primer 1 - 10-15 pmol (Final conc: 0.2-0.3 uM)
Primer 2 - 1015 pmol (Final conc: 0.2-0.3 uM)
Template - Less than 200 ng
Primestar HS Dna Polymerase - 0.5 ul
MilliQ - up to 50 ul
I run on a gel to confirm that it amplified there after but nothing. No bands. I ran my template, as is, and the band appears, which suggests to me that it is in tact.
Although the protocol suggests running the cycles as
98 degrees 10 sec
55 degrees 5 sec or 15 sec (depending on the Tm)
72 degrees 1 min/kb
for 30 cycles
But my mentor suggested running
98 degrees 10 sec
55 degrees 15 sec
72 degrees 5 min
for 20 cycles
and use 100 ng for template
He says that 30 cycles is too long and that it may lead to more background noise appearing?
If anyone can shed some light I would greatly appreciate it!
30 cycles is not uncommonly long, so I don't think you'd be getting too much background. But that said, if your mentor wants 20 cycles, he must have his reasons.
A question- how long, and what temp, is your initial denaturation step before you start with your 20 cycles?
Thanks for your reply leelee!
My initial denaturing temp and time is 95 degrees for 3 mins, and also, my after the 20 cycles, it is 72 degrees for 10 mins.
How long is your insert, 5kb?
Try run the PCR with 30 or more cycles to see if it even amplifies at all.
So you don't get band for either of the two primer pairs? Did you test the best annealing temperature for your primers? There may be 55 in protocol, but some primers may require higher temperature.
Good points, Trof.
BioChem_newbie, it is probably worth doing a touchdown and a gradient PCR to see if you can get any product at all, and to try and determine the best annealing temp for your primers. I would do these with 30 cycles.
Then if you do get a high amount of background (though I find that unlikely) you can either excise the band you want from the gel, or work on tweaking your PCR conditions to reduce background.
Some of the old taq polymearses gives high background or mutations when you have more cycles. But many of the high fidelity enzymes are much better in this regard. Also try higher annealing temp. like 60C if you have high background with 55C.
Unfortunately, we were running low on the Takara taq enzyme, so I had to switch to Rosche's kit.
I dont think its the temperature/cycle # thats the problem. When my mentor ran the same pcr/gel with the same primers, he got a good amplification band, but as I am not, I think its may be more to do with technique/pipetting issues.
I started to make a master mix whereas before I was loading each sample one by one. When I started to do that, I see bands from my mentors primers but none from mine., albeit the bands are quite a bit weaker than his, even with the same conditions. Here's where I am confused.I make a master mix for my primers and his. I use this to put into two separate 8 strip microcentrifuge tubes, so that one goes into one pcr machine for 25 cycles and the other tubes for the 30 cycle. Now theoretically, if the 25 cycle shows up for his primers, it should also show up for the 30 right...since the sample is the same (from master mix). But, the band only shows up for the 25 for his primers..... why is it that even with the same sample mix, the 25 gives something and the 30 does not?....this is where I question my technique....running a pcr can't be this stringent can it?
Thank you for any assistance!