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BYU 2014 Notebook Edit May June

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1 May 2014

Aiia: Today a plasmid prep of the Aiia plasmid was performed following the Denville SpinSmart Plasmid Purification protocol.

Alpha Amylase: Today sewing PCR was performed on the alpha amylase forward primer pieces. In order to PCR with the overlap extension primers that contain our signal sequence we will need to PCR with just the forward and reverse with the signal sequence and no template so that we have a combined forward primer to use. Then we will use that combined primer as a forward primer in a normal Q5 PCR reaction with the template. The only difference is that 2 uL of each primer should be used (the forward signaling sequence primer and the reverse signaling sequence primer). (JB)

2 May 2014

Today we ran our sewing PCR products on agarose gel and each got a product around 150 base pairs as it should have if the reaction worked properly. We then set up the next reaction with the newly sewn forward primers with the signaling sequence and the reverse primers for each gene as well as the template. We have a 5 uL control for each and then a reaction for each of the parts (including the two Amylase parts we are testing). The tube labeled Amy1 is pIG 92 and the tube labeled Amy2 is pIG98.

5 May 2014

Today we ran the gel of our PCR products of the enzyme plasmids with our sewn forward and regular reverse primers.

Aiia had a product that was around 2500 bp, where is should have only been around 800 so we need to figure out if it amplified the whole plasmid.

DispB showed smearing so either we need to play with the annealing temperatures because the primers are either not attaching at this temperature we used or the plasmid needs to be purified more.

Amy2 was the best band. This was from PIG98. We will need to digest this and the backbone and run it on low melt gel.

Left: Gel image of the sewing PCR reaction

Right: Gel image of finished gene+signaling sequence PCR products (Lane order: 5 kb ladder, Aiia, Amylase1/pIG92, DispersinB, Amylase2/pIG98)

6 May 2014

Alpha Amylase: Today I purified the PCR product from the Alpha Amylase pIG98 plasmid (Amylase2) since it appeared to have the more solid band of the two on the gel. I then prepped a vector and insert digest from the purified PCR product and the iGem backbone. Thursday I will run it out on a low melt gel in order to perform a ligation of the two together. (JB)

8 May 2014

Alpha Amylase: Today I ran the low melt gel of the psb1c3 backbone and AA insert. It turned out well and I was able to see that a segment corresponding to the length of the RFP gene was separated from the plasmid. I followed our standard ligation procedure and will allow it sit overnight in order to get a better ligation. Tomorrow I will transform it into E. coli. (JB)

10 May 2014

Alpha Amylase: Today the Alpha Amylase insert was ligated into the iGem plasmid vector and this was then transformed in DH5α following the standard procedures for ligation and transformation. (JB)

13 May 2014

Alpha Amylase: The E. coli that I transformed last weekend turned out really well. There were a lot of colonies covering the entire plate and only a few red colonies (where the RFP gene had not been excised meaning the insert did not properly work in these colonies). Today I picked eight colonies from the plate and mixed each colony that I picked into 50 uL of ddH2O, then wiped that pick on a new plate in its designated spot (numbered 1-8 to correspond with the tubes). I then boiled the tubes and the DNA acquired from these tubes will serve as template for colony PCR. I then prepped RedTAQ PCR and placed each template in its respective tube along with a control. I will run these on gel tomorrow to test the colonies in order to discover whether or not the insertion/transformation worked. I followed the standard RedTAQ procedure for the colony PCR and used 2 uL of boiled colonies as template. (JB)

14 May 2014

I ran the gel from the RedTAQ PCR yesterday and there were no bands from any of the boiled samples or control so I re-prepped a new batch of PCR and ran it. I will run the gel tomorrow. However, all of the swabs of the colonies on the plate produced good streaks. (JB)

15 May 2014

Alpha Amylase: Below is the image from the gel run to verify the presence of the Alpha Amylase plasmid in the colonies. Again, no bands showed up indicating Alpha Amylase. Today I retransformed the Alpha Amylase ligation into E. coli so that I can make more colonies. I will need to then repeat the steps of colony PCR. Desi suggested picking from 16 colonies this time for a better chance of getting a colony that will work. She also suggested using a positive control alongside the negative control, which would be the plasmid with alpha Amylase itself. This will help ensure that all the reagents are working. (JB)

19 May 2014

Alpha Amylase: Some good news finally - I froze down my DH5α + ligation transformation from last time so I was able to save some time and just replate some of that. Tomorrow I will pick from colonies and do a 16-colony PCR to see if the gene was inserted into the DH5α. Dr. Grose said it was likely issues with PCR that caused the lack of bands last time since many of the colonies were no longer red last time which indicates that the transformation did work. (JB)

20 May 2014

Alpha Amylase: The E. coli colonies from yesterday turned out really well. There were not as many colonies as last time but there was only one colony that I could see that was a pinkish color. I prepped colony PCR from 16 different colonies which I labelled on the plate. I then used those picks as template and streaked them in their respective quadrants. I will run the 16 colonies and control tomorrow on gel after PCR. (JB)