Since all the reactions yesterday worked, I purified the DNA and took some measurements in the nanodrop:
- Tube 1: 289.6ng/ul in 30ul –> 8688ng
- Tube 2: 259.8ng/ul in 30ul –> 7794ng
Since those yields look phenomenal, I decided to run my digestion of the anchor with BstXI. Once I gel extract my pBR322 digestion, I can run the final step, LIGATION! I’ve never completed this process in 2 days, and I hope I didn’t just jinx myself. Anyway, here is the digestion of pALS:
I took the following gel image on my phone and used photoshop express to enhance the contrast. All gel reactions worked. Note the smudge is a reflection of light off my filter on the illuminator.
I’m going to try to make more unzipping DNA in one final push for my PhD. Here goes nothing, I’m all in now!!!
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:
- 1kb ladder
- 1mM MgCl2
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!
Time to optimize the PCR reaction so that I can be sure the reaction will work every time efficiently. This way I can just do a few more PCR reactions and give myself a good supply of 4kb anchor to work with from here on out. Here is the reaction:
Well both reactions from yesterday failed. I’ll have to do a Mg++ titration to see if I can optimize the reaction at the original temperatures.
In the meantime I do have enough to move on and go to the digestion and ligation reactions. Moving on…
I’m making slight modifications to the last reaction before I move on:
- I’m changing the ThermoCycler program temps just a little bit based on some observations I made with the machine yesterday. I noticed that running the machine on manual isn’t the same as running a program, during a program the block actually will go over (or under depending on the situation) temp while the machine measures the temp of a tube with oil in it. My measurements from yesterday did not experience this in manual mode.
- I’m also trying OneTaq in OpenPCR with some slightly modified temps, hoping that helps the reaction a little bit.
Here are my reactions: