I have finally received my gBlock shipment from IDT. A gblock is guaranteed to be 200ng, which is a little nerve wracking to work with- it is not a small amount of DNA, but it is not a huge amount of DNA either. A colleague at BOSSLAB suggested that I add PCR priming sites to the end of the synthesized DNA, to let me amplify it before diving into cloning. This post explains how I amplified it, and what kind of yield I got, and how I tested that I got the “right thing”.
I chose to add standard 17-mer universal primers to my sequence, so along with my gBlock, I got two tubes of primers. I chose to use Q5 polymerase because it is the highest fidelity polymerase that NEB sells. For a “typical” PCR reaction, NEB recommends re-suspending primers to 10uM, and using less than 1000ng for template. Since all my DNA was shipped dry, I needed to first resuspend my samples to the proper concentration, then run PCR and verify that I had made the correct product.
IDT conveniently tells you how many nanomoles you have been shipped. To get a 10uM concentration all you have to do is add as many ml as you have nanomoles, since 1 nanomole per milliliter is 10uM. So, after a brief (5 seconds or so at 5k) spin to make sure all the dry DNA is at the bottom of the tube, then I added some water, at 1ml per nanomole. Unfortunately, I had 3 or so nanomoles- and the tubes can barely fit 2.5 ml. So I resuspended in 1ml of water, split the resuspended DNA into an extra tube, and then brought both tubes up to the correct 10uM concentration. IDT ships you a lot of primer if you order custom oligos.
Resuspending the gBlock was a little scarier. Unlike the primers, the gBlocks are not visible in the tube (as far as I can tell). So after a quick spin, I shot 20ul into the bottom of the tube and pipetted up and down to mix.
With everything resuspended, it was time to set up the PCR! Fortunately, NEB has awesome protocols on their website, so it was easy to set up. The Q5 even comes in a 2X master mix, so it was easy to pipette! I made two 50ul reactions:
- 25 ul 2x Q5 poly master mix
- 2.5 ul m13 forward primer (10uM)
- 2.5 ul m13 reverse primer (10uM)
- 1.0 ul template DNA (10ng/ul)
- 19 ul PCR water
The PCR program was run with a pre-heated lid (114C) and a hot start (98C). The annealing temperature was determined with the NEB Tm calculator. I ran 30 cycles of:
- 30s detnaturing at 98C
- 15s annealing at 57C
- 45s extension at 72C
There was a final extension of 72C for 5 minutes, and then a final hold at 4C.
A 1% agarose gel with 5 ul of 20,000X “Safe DNA Gel Stain” (from bioland scientific, via the odin) was prepared. Four lanes were run- 5 ul of 2 log ladder in purple dye, PCR product 1 and 2, and then 1 ul of 2 log ladder in purple dye. The PCR product was expected to be 1k3 bp, and you can see it right in between the 1.2 and 1.5 kb bands in the 2 log ladder.
For those interested in how sensitive the DNA stain is- those bands on the bottom are from NEBs 2 log ladder. From left to right, the first band is about 40ng/ul. As you know, the top band has 5 ul of ladder loaded, while the bottom has 1 ul of ladder. That means this is a photo of the position of 40 ng of DNA. I was impressed by the sensitivity, especially because this was not shot under ideal conditions- I did this in a dark corner of a room, under the shade of some paper I found lying around. With a better setup, I think the 40ng bands could look even better!
The very observant reader may notice some faint bands in the second lane from the top, which correspond to the ladder on the top. This gel tore across the lanes when I separated it from the casting apparatus, and I think some of the top lane leaked into the neighboring lane. Fortunately, the PCR products are in a different position and at a different intensity than any of the ladder bands, and UV spectrometry verified that there was DNA that was amplified.
DNA Quantification by UV Spectrometry:
While we do not have a nanodrop, we do have a genequant. Just like a nanodrop, it is a UV spectrometer that is specifically designed to quantify DNA/RNA in samples. Unlike a nanodrop it requires at least 50 ul of sample for the cuvettes that we use. Since the DNTPs and proteins left over from a PCR reaction will also absorb UV wavelengths, and because PCR buffers and enzymes can have non-specific and unwanted activity later on, there is such a thing as a “PCR cleanup kit”, to extract out the product of your reactions. Unfortunately, this kit will also concentrate the reaction products into smaller volumes, around 25 ul. I combined the two samples eluted from the cleanup kit and measured them in the spectrometer, which told me I had .071ug/ul, or 3.5 ug total. That is about a 350 fold increase in DNA! I thought that was pretty good.
With PCR product in hand, the next steps in cloning involve cutting, de-phosphorylating the backbone, and ligating the insert into the backbone.