Do you know that feeling you get when you realize something that you did, that you know you weren’t supposed to do, but didn’t catch it until it was too late? I had a few of those this week. They were simple mistakes, but they are also the reason that we haven’t been getting as much growth on our plates as we would like. I had previously mentioned that when you run a PCR, you must also clean it afterwards to remove all of the primers and other junk that you put in with the plasmid. When setting up a ligation in order to grow colonies on plates, you must use this cleaned PCR in your reaction. This brings us to our first mistake; I have not been using the cleaned PCR. This explains why we have not been having much growth on our AS1/AS2 plates. It is hard for colonies to grow with all of this other “junk” mixed in with the PCR.
Mistake number two: When setting up the “uncut” plate during a ligation, I have been using the plasmid that was digested with a restriction enzyme, meaning that the enzyme I used has cut the plasmid. You would think that the word “uncut” would tell me that I should not use the plasmid that has been “cut”. After resolving this issue, this is how our “uncut” plate looked.
Other than fixing my mistakes this week, we also focused on determining the orientation of our AS1 clone! In order to do so, we took two different approaches. To begin, we drew out what our vector map (pET-23a), including how many base pairs the vector consisted of, and found restriction enzymes that “cut” the vector multiple times. In a normal digest, we use BamHI, which is a non-cutter. Because it does not cut the vector, we only see one band on the gel (the plasmid). For an enzyme that cuts once, we would see two bands, two cuts we would see three bands, etc. The two enzymes that we chose to digest were XhoI (1 cut) and PstI (3 cuts). Below is a picture of the vector map that I drew, including symbols such as B (BamHI), X (XhoI), and P (PstI) for where the enzymes cut on the vector. Along with each symbol, the base pair number in which they cut are also present. From B (198) to B (230) is where the AS1 insert would be. Each “cut” is color coordinated on the map to show how long (how many base pairs) the cut should be.
When we run the gel in this case, we would expect to see 4 bands for the PstI digest (bands being at around 32 base pairs, 822 base pairs, 198 base pairs, and 2,546 base pairs), and 2 bands for the XhoI digest (bands being at around 4,310 base pairs an 428 base pairs).
The other approach that we took in order to determine the orientation was to set up a PCR reaction using the AS1 clone as our template DNA. In a normal PCR reaction, you have 1 microliter of your template DNA, 1 microliter of a reverse primer, 1 microliter of a forward primer, 12.5 microliters of your PCR master mix, and however much water is needed to bring the reaction up to volume. In this case, we set up three different reactions: one being our positive control by using the normal AS1 forward and reverse primers, one being, hopefully, our negative control by using AS1 reverse primer and a T7 Reverse primer (if there is a band on this reaction, it shows that our clone is in the wrong orientation), and the last using AS1 reverse primer and a T7 promoter (this being where we hope to see our band).
We ran both the PCR reactions and the digests on the same gel below:
In lane one (the very bottom) is our marker, in lane two we have our PstI digest (3 cuts), in lane three is our XhoI digest (1 cut), in lane four is our positive control PCR, in lane five is our negative control, and in lane six is our AS1 reverse primer and T7 promoter.
For the PstI digest, there are three bands present (because the 32 base pair band is so small, we do not expect to see it on the gel). For the XhoI digest, there are two bands present, both appearing to be in the correct locations, showing us a positive sign for the clone to be in the correct orientation. As for the PCR products, there is a band for the positive control, and one for the AS2 reverse and T7F primer, which is a good thing! Because there is no band on the negative control, more than likely our clone is in the correct orientation! Next week, we will continue prepping our clone for sequencing, and we will prepare our AS2 plasmids to determine if the DNA insert is present.
Also this week, we transplanted our wildtype, AS1, and AS2 plants into soil!
Left: Wild Type Middle: AS1 mutant Right: AS2 mutant