April 23, 2013 in Posts
We have successfully shown that we are able to barcode an unknown fungal sample, but the question now is, can we reproduce it? We have gathered the necessary tools and now face the task of making sure that our protocols are thorough, easy to follow and most importantly, reproducible. Over the past week we have ran ~28 reactions (DNA extraction, PCR, isolation). Unfortunately, what we have found is that our protocol is not as robust as we were hoping.
Samples 1 to 3 are DNA extractions from the same fungal colony using 3 different DNA extraction solutions. All used ITS primers (see below for primer sequences).
Sample 1 used an extraction solution of Tris-HCl, EDTA and SDS.
Sample 2 used 20mM NaOH
Sample 3 used 20mM KOH.
Samples 4 and 5 are DNA extractions from the same plant sample using Epoch Life Sciences Plant Genomic DNA Extraction Kit (Cat # 1560050).
Sample 4 used primer set LEP
Sample 5 used primer set rbcLA
Sample 6 used primer set rbcLA and a plant template extracted using 80mM NaOH
Sample 7 used fungal DNA extracted using 80mM NaOH and ITS primers
So what do we think is going on?
As for Sample 1 , we initially thought our extraction solution would be effective for lysing cells and extracting gDNA. Unfortunately, at the time we were not thinking of the impact SDS and EDTA would have on the polymerase/overall PCR reaction. SDS is a strong detergent that is often used to lyse cells and extract DNA, but is also very good at unfolding proteins. That is not good news for our polymerase. The other problem with sample 1 was the EDTA. Metal ions (specifically Mg2+) are essential co-factors for our polymerase to work correctly. Having too little or too much free Mg2+ can be devastating to a PCR reaction (as we are finding). In our case, the presence of excess EDTA and SDS rendered our reaction, unreactive…
Sample 2 and 3 seemed to work very well with 20mM NaOH and KOH, respectively. The simple colony PCR protocol was a tip given to us by an experienced molecular biologist. We figured that the base would be aggressive enough (but not too aggressive) to lyse cells and make DNA available for extraction. We were right! It felt good to know that that method worked since it’s so cheap and fast.
Sample 4 was expected to fail as the primers were designed for insect DNA amplification.
Sample 5 worked as anticipated. Again, this sample used the Epoch kit, so started with clean plant genomic template DNA.
Sample 6 was a plant prep using 80mM NaOH. It was encouraging to see enough plant DNA could be prepared by such a simple, fast, and cheap method and give comparative amplification to Sample 5, which was prepared using a spin kit costing ~$1.50 per prep.
Sample 7 was a failed ascomycete fungal sample prepared by grinding the sample in 80mM NaOH.
Standard 25uL reaction
1uL template DNA
0.5uL 10uM forward primer
0.5uL 10uM reverse primer
12.5uL NEB Taq 2x Master Mix
Overall, these 7 samples showed us that out procedure was somewhat reproducible, but we do have some variables to consider looking into. Primer design and PCR conditions for each primer set are definitely worth checking out. Also, maybe our Taq is starting to go bad; we have taken it in and out of the freezer dozens of times, same goes for our primers. We will definitely look into this further.