• Midiprep pEXPR_PirBTCSG4VP16. The midiprep concentration was low, around 16. Probably because the culture didn't grow very well.
  • ^Miniprep inoculating again. Also, the 3rd floor shaker temperature sensor is busted.
  • Troubleshoot PCRs with Brian / there is nothing we can do. Sadface. Design new primers or such? 
  • Digest any YFP fusion minipreps we have to check for what segments are present and what went wrong.
  • Dilute midipreps.


Things we have left:Problem
pEXPR_hEF1a:LilrB2_TCSG4VP16LilrB2 PCR not working; TCSG4VP16 PCR is fine
pEXPR_hEF1a:LilrB2_YFPLilrB2 PCR not working; YFP PCR not working
pEXPR_hEF1a:PirB_YFPPirB PCR is fine; YFP PCR not working

Lab work left: Troubleshoot PCRs. The rest should be fairly simple.


  • I may have used the wrong primers to do PCRs yesterday. Need to check for that.

Nope. Used SS13 and SS14, which are the LilrB2 YFP PCRs.


Debugging: 

 

  

Look at the first lane of LY. That tiny piece means that the rear half of LilrB2 is there. This means that the reverse primer worked. But it gave us a smaller band yesterday. What is the meaning of this?

 

 

Digested 5 samples of pENTR_LilrB2/PirB_YFP to check which inserts are actually present. It should help to clarify which PCRs are the biggest problems.

 

The geneious files are in Sandboxes>Shinjini>LilrB2,PirB>pENTR_LilrB2/PirBYFP copy

I won't be changing the copy files, so you can take a look at where the enzymes cut and try to figure out which parts are missing.

 

 

 

Looking at LilrB2, it looks like all the XbaI's cut. That means that the LilrB2 gene is in all of them.

For PstI, the PstI cutsites are on the back end of LilrB2, the section that missing for LilrB2-TCS. I'm guessing that the same thing happened for this LilrB2. Looking at LilrB2_YFP 15 (the first one), the small band means that the LilrB2 may have extended enough to have at least one of the PstI sites. I think the YFP is there.

I think it's worth sending it in for sequencing.

PirB wise:

3rd one is obviously missing the PirB gene. It have the YFP, though.
The others have the PirB gene. So, the top band is the cut at the PirB gene. That would mean that the second bands are because of YFP. But that would mean the section between the cuts are absurdly large. Comparing it with the ClaI cuts, the total of the two bands in PstI would be MUCH bigger than it is ClaI, which is impossible. Or, the top band is a single cut and the second band is a double cut with a really small band that's not visible.

I think it's worth sending PirB PY20 (assuming it's representative of the others) for a forward and reverse sequencing.

 

 

8/11 comment: I had confirmed that the pENTR_LilrB2 we are using is fine (L2 and L3). The sequencing primer in the middle didn't primer, but that's probably because the primer preparation was faulty, and it had primed before. Note to self and everyone: ALWAYS take all precautions before moving on, it saves a lot of effort. And ALWAYS document everything. Especially gels.

I think the pENTRs should be fine, but I have no logical explanation for the fusion digestion band patterns, and the fact that the LilrB2TCS sequencing showed that the rear end of the LilrB2 gene was present.

8/12

This is the section that's missing.

If you look at the sequencing results, the first 10% (containing XbaI) belongs higher up in the gene. The rest of it belongs as shown.

It's missing the NcoI site, so if we test cut there for the original pENTRs, we should be able to tell if they are missing anything...


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