September Notebook

9.12.14 Digest of minipreps to check for inserts

Background

Making sure the inserts are here from the colony PCR results - are the GFP and RFP inserts present?

Digest each plasmid with EcoRI/PstI. Incubate 15' at 37C.

Run entire digest on 0.8% gel.

Results

These were slightly confounding. What I *think* happened is that the buffer wasn't optimal for one of the enzymes, so the digest wasn't run to completion. Why do I think this?

The RFP plasmid should cut to 2.1 kb (backbone) + 0.8 kb (insert). In plasmids 3 and 9, this is the case.

In the rest of the RFP plasmids, the linearized plasmids appear to be nearly 3 kb. This makes sense if the insert is there but one of the enzymes didn't cut. If the insert weren't present, the backbone should be the 2.1 kb only. Thus, I think they *all* actually have the insert, but we will work with the two confirmed plasmids.

Conclusions

Move forward with RFP 3 and 9.

9.26.14: Digestion to prepare for part verification

Background

We need to verify that the fluorescent proteins are fluorescent in the proper color. To do this, we have to first clone the gene (which already has an RBS) into a plasmid with a promoter. This will allow us to express the genes in E. coli for further verification.

Set up digests:

For each fluorescent gene plasmid (OFP, CFP, and RFP. No GFP bc digest looked funny on 9.12:

Digest the plasmid with EcoRI and PstI.

Digest pUC18 with EcoRI and PstI as well. pUC18 is a plasmid with a T7 promoter

Incubate plasmids for 60 min at 37C and then HEAT INACTIVATE enzymes for 20 min at 82C (This last step is very important because we want to use the products of the digests without further purifying them, and we don't want the restriction enzymes to be active when we are putting together the ligations!!!)

Results

Plasmids are digested and ready to use!

9.26.14 Antarctic phosphatase treatment of pUC18

Background

We are going to treat the backbone with Antarctic Phosphatase, the protocol in this case is:

1. to the vector tube (which contains 20ul digested pUC18), add 5ul of 10x Antarctic Phosphatase buffer, 25ul of water, and 1 ul of Antarctic Phosphatase. We do this to prevent the vector from closing up again without any insert.

2. Incubate the tube at 37C. After place at 82C for 20 minutes.

Results

We assume that the DNA is fully dephosphorylated and that the phosphatase enzyme is denatured.

9.28.14 Transformation of ligation products into E. coli

Background

Transform 5 ul of each ligation product into commercial chemically competent cells. Incubate at 37C overnight.

Results

Look great! Clearly some colonies are fluorescent and some are not (see image)

Conclusions

Next step: purify the proteins from the cells to ensure they are fluorescent at the correct wavelengths!