I had to split each reaction into 2 lanes because the volume of the reaction wouldn’t fit in 1 lane.
So this reaction failed. I’m guessing the digestion of pALS didn’t work. There is an interesting artifact in this gel though. In lanes 2 and 3 you can see 2 bands between 2 and 3 kb. I have no idea why the DNA would separate like that. There are literally only 3 pieces of DNA in here: a ~30bp piece which wouldn’t be visible in this gel, a 2.5kb piece which is the EpBR fragment and is where the 2 bands are, and the 4kb pALS piece which is above these dual bands.
Oh well, I’ll try again at a future date when I run out of the DNA that I have currently produced.
Hooray! While the gel image isn’t the best picture ever (thanks phone), the good news is that the PCR reaction worked for every MgCl2 concentration and seems to work best at 3.5-4.5mM. Also it should be noted that lane 3 contains a visible band, but there was some smudge on the filter so it blocked the light from that lane. Here are the lane assignments:
I have purified the reactions and according to the nanodrop, I have about 11ug of DNA which translates to 87nM (233ng/ul) of tetherable DNA.
I found some EpBR (EarI digested pBR322 and gel extracted) so I’m going to digest some of this new pALS DNA with BstXI, purify, and then ligate with both adapters. If all goes according to plan, I’ll have all the unzippable DNA I’ll ever need!
It looks like the ligation didn’t work exactly as planned, but I would say definitely better than the 3 piece ligation debacle. I also let the gel run for too long, so the 1, 1.5, and 2 kb bands have run off the gel. The bottom band in the gel is the 3kb band. The brightest bands in my gel are the 4.3kb linear pBR322 fragment and then what would be the ligation product. And it appears the ligation didn’t work at all in the 5’bio reaction from this morning. Oh well gel extraction and unzipping attempts are next on the “To Do.”
So the good news is that had I not thoroughly messed up the reaction, this would have been a resounding success. The other good news is that this part of the experiment is fairly simple to repeat using the pRL574 anchor. The bad news is the ligation went as I expected it to which is to say sloppily.
Now I have to try to explain to you these results:
Above is an image of the gel under UV light stained with Ethidium Bromide. I used to use Sybr Safe for visualization (and have in the first part of these experiments), but in the past unzipping was never successful so we think there is a chance that Sybr Safe interacts with DNA differently than EtBr and could hinder unzipping. So in an effort to identically repeat Koch’s grad school work I’m using EtBr because that is what he would have done.
Below is a cropped and grayscale version of the above image:
We are looking at a lot of lines here. There is a meaning to them all though:
The two outer lanes are 1kb ladders in different amounts. More specifically the right most lane has twice as much ladder as the left most lane. We’ll call these lanes 1 (left) and 5 (right).
The three middle lanes from left are: ligation with 5′-bio adapter (lane 2) and pRL anchor, internal-bio adapter and pRL anchor (lane 3), and 5′-bio adapter with pALS anchor (lane 4).
The ladder reads (from the bottom): 1kb, 1.5kb, 2kb, 3 (brightest band in the ladder), 4, 5, 6, 8, 10kb
Lanes 2 and 3 are identical length wise and the only difference between the molecules is where the biotin molecule is located.
The band at the very bottom is the pRL574 PCR product band which is 1.1kb in length.
The next two bands above that are pBR322 that has religated into circular DNA.
The band above that is digested pBR322 (4.3kb).
And the band above that is the unzipping DNA that we want, which is around 5.4kb. I have gel extracted that piece.
All bands above this band are chains of pBR322 that ligated together and may or may not have the anchor and adapter ligated to it.
Lane 4 is interesting. I would be inclined to say that we got better ligation out of this band because there is less circular DNA bands (compare with lanes 2 and 3). But the product is harder to quantify.
The brightest band is the pALS anchor, 4kb in length.
Barely above that is the digested pBR DNA, 4.3kb in length. And is identically positioned with the other two lanes.
Above that is a very feint band and now I’m inclined to believe that this band represents two pBR322 fragments ligated together and circularized. (Above I say its the product of the reaction that I wanted). I think this because this band lines up exactly with bands in the other two lanes.
The next visible band I believe is the product that I expect, 4kb pALS + 4.3kb pBR -> 8.3kb unzipping sequence. It is right there, but again bands in the other lanes could indicate something else.
Well tomorrow I’ll gel extract and hopefully we can get some DNA tethers by the end of the week and try for unzipping. Also tomorrow I’ll retry the ligation reaction for pRL-pBR (since I have no more predigested pALS). Tethering is the only real way to identify successful ligation so we’ll find out together!
I ran a 0.8% gel prestained with Sybr Safe and viewed with the invitrogen illuminator for this stain.
And below is the image of the gel taken with my crappy-ass camera phone (Droid Bionic).
Yay! The reaction finally worked! Well mostly. It worked in lanes 6-10, which correspond to one of the reactions from the OpenPCR and 4 of the reactions from the ThermoCycler. I will assume the 5th reaction worked as well. That reaction is not visualized because there are only 10 lanes in the gel and there are 10 reactions plus the DNA ladder so one would need to be left out.
I did a reaction cleanup with Novagen PCR Cleanup. I couldn’t find my Qiaquick PCR cleanup kit and this was all I thought I had. After cleanup, I found my Qiaquick kit and will use that next time.
The nanodrop says there are 35.3ng/ul of PCR product which correlates to ~13nM. That isn’t great but it’s a start. Moving on… FINALLY!!!
0.8% gel: 50mL of 1x TAE, 0.4g agarose, prestained with Sybr Safe
out of 10 reactions only 1 reaction worked… hmmm. And there is a chance that the product isn’t even what I want. It appears that the band is not 4kb, but the gel didn’t run evenly so there is a chance it is 4kb but doesn’t appear that way because of how the current pulled the DNA.
It looks like a machine test is in order. The Thermo cycler said the program completed today (which it hadn’t in the past few trials), but also said the program completed in 2.5 hours (which it shouldn’t because it is a 4.5 hour program). And the OpenPCR won’t get below 16C for the final hold even when I set it at 15C, which it should not have a problem holding at (I’ve seen it consistently get to 12C).
I will also order new dNTPs even though I think that may not be the problem.
I also learned yesterday that the freezer wasn’t closing completely and everything may have thawed potentially ruining the enzyme.
Finally I should try to mix the master mix better if that is an issue at all.
Lots to do in so little time… Gotta get this working by this weekend.
Blah! I grow disdain for failed PCR reactions. I have no idea what the issue is and I hate troubleshooting PCR. Oh well. I’ll stop venting and get right back to work.
So the PCR was a failure again. And now I have to figure out how to adjust it. Since I’m not getting any product here are my potential fixes:
adjust the annealing temperature
try different amounts of MgCl2
try new dNTPs
In order to properly adjust the annealing temp I would need to make sure the machines are functioning properly, which I’m figuring they aren’t. The reason I think this is because when I check the thermal cycler (not OpenPCR) it says Program end in 0:00 and usually says Hold 4C. But the reason I think the thermal cyclers are not the issue is because neither of them produce product, their reactions are identical, and their temps are very similar.
In order to troubleshoot the machines I’ll have to run a temp experiment where I track the temperatures over time. I have equipment for this, but I can’t find all the components. I’ll have to talk to Pranav about the missing parts.
Trying different amounts of MgCl2 is easy. I would just do amounts ranging from 1 to 5mM of MgCl2 all on the same program.
In order to try new dNTPs I would just need to buy new dNTPs. This can’t happen until Monday. Neither can the thermal cycler experiment. Tomorrow I can try a MgCl2 titration experiment. Right now I’ve got a simple check in mind.