Category Archives: PCR

PCR yield and BstXI digestion

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:

PCR reaction results – Succesful

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.

20130123-134830.jpg

pALS PCR 7 (titration) results – SUCCESSFUL

image

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:

  1. 1kb ladder
  2. 1mM MgCl2
  3. 1.5mM
  4. 2mM
  5. 2.5mM
  6. 3mM
  7. 3.5mM
  8. 4mM
  9. 4.5mM
  10. 5mM

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!

pALS PCR 7: Mg++ Titration

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:

pALS PCR 6: results

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…

pALS PCR 6: setup

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:

pALS PCR 5: results – SUCCESSFUL

Here is the setup from yesterday.

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).

image

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!!!

Thermo PCR Sprint (“ThermoCycler”) Programmed T vs Recorded T

This experiment is a follow up to yesterday’s experimental results. Instead of trying to program the ThermoCycler by guessing, I decided to record the temperatures of various different T settings. Basically I would pick a T and try to get the recorded value near the T’s needed for the pALS PCR protocol.

I put the machine in manual mode so I can change the T when I needed to and I would take data points at 30 seconds for melting and annealing temperatures and at 30s and 5 minutes for extending T. This is how long I would be doing that step during the PCR reaction so it seemed to make sense. Below is a chart of the Set T and the recorded T at each interval.

Below that is the data of the recorded T.

The recorded temperatures of the PCR Sprint thermal cycler.

Figshare Data:

PCR Sprint Programmed T vs Recorded T. Anthony Salvagno. figshare.
Retrieved 18:51, Aug 23, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.94414

pALS PCR Try 5

SO I’ve done the temp experiments and decided to do a quick temp reading of the Thermo Sprint PCR machine this morning. That data will be up shortly. The temperatures for the PCR reactions below are based on those readings, while the T for the OpenPCR reaction (also below) are based on the readings from the experiments the past few days.

Anyways now I have two machines that are better prepared to perform this PCR reaction. Both machines are set for actual temps of ~92C, 50C, 72C and hopefully I get something that I can work with today. Reaction setup is here: