PCR Amplification Issues and Primer dimer - (Jun/29/2015 )
Hi all,
I have been working on getting my PCR done correctly for months with no progress. Most of the time I just get a primer dimer of about 100 bp when run next to a ladder, as my sample is about 360 bp. Every now and then I will get a really nice amplification but these results are not consistent (actually I've only got 3 good amplifications in the past 5 months). I will start from the beginning:
Extraction:
I use a modified protocol developed by former lab mates. I typical extraction yields around 10-100 ng/ul of DNA with relatively little contamination (or so I think; 260:280 is consistently around 1.8), although I've been told that polyphenolics could be inhibiting the PCR (I have not really looked at this yet).
PCR:
In my lab we are amplifying the Internal Transcribed Spacer 2 Region of Symbiodinium (Dinoflagellate) DNA and parts of the 5.8S region as well. The extracted DNA is amplified through PCR using Touchdown PCR and eventually run on DGGE.
My problem:
Former lab mates have developed a "recipe" for PCR that has worked consistently for the past 13 years. I can provide more information on the primers if needed.
Recipe (per reaction; concentrations are initial not concentration in reaction):
16.7 uL Water
2.5 uL 10X Taq Buffer w/o Mg
2.5 uL dNTP (2.5 mM each)
1.0-2.0 uL Mg (25 mM)
0.25 uL F primer (10 uM)
0.75 uL R primer (10 uM)
0.13 uL Taq
1.0 DNA (usually diluted 1:10 or 1:20, but have tried undiluted and dilution series)
This recipe is not working for me but it should be working. I have replaced all the reagents thinking that they had gone bad, but still no luck. The reverse primer used in my PCR has a GC clamp which I have read can cause issues with primer specificity however we use the touchdown PCR to increase specificity. I have tried using the same reverse primer without the GC Clamp and had amplification but these results were not reproducible, even when using the same DNA that was successful. I have also tried additional primers that other lab mates use (they amplify the same area and an additional area) and they do not work as well. About 90% of the time I just get primer dimer with not even the slightest band. The other 10% I do not even get primer dimer. I have also tried using two different programs on multiple thermocyclers.
Thermocycler: Using Touchdown PCR these are our following temperatures (I have tried two programs)
First program:
92C (2 minutes)
Denature 92C (30 seconds)
Annealing 58C (40 seconds)
-0.5C per cycle (24 cycles)
Extension 72C (30 seconds)
Repeat for 24 cycles
Denature 92C (30 sec)
Annealing 52C (40 sec)
Extension 72C (30 sec)
Repeat 18 cycles
72C (10 minutes)
Hold
Second program:
92C (2 min)
Denature 92C (30 sec)
Annealing 62C (40 sec)
-0.5C per cycle (20 cycles)
Extension 72C (30 sec)
Repeat 20 cycles
Denature 92C (30 sec)
Annealing 52C (40 sec)
Extension 72C (30 sec)
Repeat 20 cycles
72C (10 min)
Hold
I am beginning to think that it may be problems with my DNA extraction but I would like to ask if anyone sees any issues with my protocols or have any suggestions to try. I forgot to mention but I have tried a Nested PCR but that has also failed. Thank you for taking the time to read and any insight would be greatly appreciated.
Cheers,
D
I would be doing the denaturing at 94 degrees with Taq, both initially and in the cycle. Also, both cycle conditions use a shorter than usual number of cycles. I would do 34 cycles, and touch down more gradually -- 0.2 or 0.3 degrees per cycle. I would also try just a 55 degree fixed annealing temperature.
The small number of cycles probably is the biggest issue.
If you have a gradient cycler, I would try gradient cycling like this:
94/60s
then 34 cycles of:
94/30s
gradient 48-66 degrees/30s
72/30s
then 72/10m
You can do this reaction by making 100 ul reaction (double what you make above) and aliquoting 12 ul in each of 8 wells of an 8 well strip.
There might also be an issue with your gDNA prep. How is it being done?
By starting with such low annealing temps you are defeating the purpose of touchdown PCR. It is important that you start with high annealing temps to build up the specificity, then work lower.
My favorite TD protocol, which pretty much always works for any traditional amplification from plasmid or gDNA...often times using primers with predicted annealing temps 10-20C different (e.g. sequencing primer with mutagenic primer) is:
95C 2min
95C 30 sec
68C annealing 30 sec
-1C per cycle
72C 30 sec per kb
repeat 12 times
95C 30 sec
55C annealing 30 sec
72C 30 sec per kb
repeat 25 times
72C 10min
4C hold
Other things to consider:
Why are you using different concentrations for forward and reverse primers?
When you set up your reactions, are you actually trying to pipette volumes less than 0.5ul? Or are you making a master mix before hand? I would highly suggest the latter as pipetting less than 0.5ul is most unreliable. This could explain some of the inconsistent results.
Hi labtastic
I was told that we use more of the reverse primer because of the low specificity that is a result of the GC clamp. I have tried bringing it down to twice as much but it does not work. When using the same reverse without the GC clamp we use equal amounts.
I make a master mix, but that is the amount we designate for each reaction.
I will try this protocol today. Thanks.
Cheers,
D
Hi phage434,
I do not have a gradient cycler, but I'll see if someone else does and get back to you.
As for DNA extraction, it is just a modified Promega Wizard kit that someone in the lab developed a while ago.
1. The cells are washed and pelleted.
2. Proteinase K, Nuclei Lysis Buffer, and glass beads are added and placed in the bead beater.
3. Incubate at 65C for 1 hour
4. Add RNase
5. Add 9M ammonium acetate
6. Supernatant, 100% ethanol, and 3M sodium acetate added
7. Spin down and pour off
8. Wash with 70% ethanol
9. Dry for at least 3 hours in hood
10. Resuspend pellet in 100 uL water
There are also many more steps, but these are the main parts - I can upload a copy of the protocol if you would like to see it.
Cheers,
D