Hello all, I am trying to do my first successful PCR reaction and I am on my 8th attempt, and still have gotten no bands. This is just a control reaction using plasmid templates which should contain the matching primer sequences. Most of the info here is the details of my reagents and at the end is my master mix ingredient list and PCR protocol. Any help would be really appreciated.
--- Plasmid Templates:
I am using mainly freegenes plasmids, which are mostly biobrick parts and it seems every single one has M13 sequences flanking the genes. Here is an example plasmid with it's alleged sequence (one of the ones I am using as a template): gfasPurple_BB3335
I have been miniprepping the plasmids from e.coli dh5a getting 20-100 ng/ul concentrations using neb miniprep kit and eluting with neb monarch elution buffer (which is supposedly TE buffer).
--- Primers:
First, I ordered "readymade" m13 primers from IDT specifically the "m13 forward (-20)" (GTA AAA CGA CGG CCA GT) and "m13 reverse (-27)" (CAG GAA ACA GCT ATG AC). Both are 17 bp long. They came as a pair of seemingly empty 2ml tubes with 10ug of primers in each tube which I then pipetted 100ul DI h20 into and verified ~100ng/ul using nanodrop. I calculated this to be ~20uM each based on ~5200MW on each primer.
--- Taq Kit:
I ordered neb taq pcr kit which includes 10x standard taq buffer, 5U/ul Taq DNA Polymerase, and 10mM each dntps solution. The whole kit I have been keeping in a -20c freezer.
--- Water Source
The DI water I am using for my reactions is a cheap filtration kit just like this one. It includes a sediment, carbon, and block filter, reverse osmosis membrane, and mixed bed deionized filter. I read elsewhere that you can do PCR with tap water and so I figured this was sufficient, but I've never done PCR before so I actually have no idea.
--- Procedure
Okay onto my procedure. I first diluted my plasmid extractions to ~ 1ng/ul to serve as templates. These should supposedly have m13 flanking sequences, and I add 2ul of this to each reaction tube for a total of 2ng of plasmid DNA. I setup my master mix as below, which is based on neb's taq protocol as a guideline.
> 10x standard taq buffer: 20 ul
> 10mM DNTPs mix: 4 ul
> 19uM m13 fwd primer: 2 ul
> 19uM m13 rev primer: 2 ul
> 5U/ul taq dna pol: 1 ul
> DI H2O: 165 ul
Total 194 ul master mix, which leaves 6ul of volume for template DNA addition. Final reaction vessel mixtures are below, put in standalone 0.2ml PCR tubes.
A: 2 ul plasmid1 (~2 ng), + 48 ul master mix
B: 2 ul plasmid2 (~2 ng) + 48 ul master mix
C: 2 ul plasmid3 (~2 ng) + 48 ul master mix
D: 50 ul master mix negative control
The thermal cycler I am using is a MJ-Research PTC-200 "DNA Engine" with Dual Block head. I set the protocol to use a constant temp heated lid to 60c and temp control method to "calculated" based on 50ul reaction in PCR tubes. The program was set to the following:
1: 95c 1min
2: 95c 30s
3: 47c 30s
4: 68c 1min
5: loop 2-4 29x
6: 68c 5min
7: 10c hold
The anneal temperature of 47c was chosen based off of neb's Tm calculator and given the m13 primer sequences and final primer concentration of 200nM. The extension time is based off of neb's pcr protocol guideline of 1min per kb of target segment length, the target sequences from the plasmid maps for these plasmids are around 700-1000 bp.
--- Final Notes:
There was considerable condensation near the lid of the tubes at the end of my last reaction, I don't know how much of an affect this would have.
I have altered the above protocol a couple of times trying 50 and 55c anneal temp and 40s anneal time. I have tried a second taq kit in case my first one had a bad reagent. So far I have not used a different set of primers so that is my next thing to try.
I believe I have given all the info I can think of. At this point I have no idea why I am not seeing bands and not sure what to try next. Once again any help or advice would be very appreciated.
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