Team:BYU Provo/Notebook/Cholera - Enzyme/July-August/Period2/Dailylog

From 2013.igem.org

Cholera - Enzymes Notebook: July 15 - July 31 Daily Log

Cholera Enzyme

March-April

7/15/13

We poured a low-melt gel for the Savinase and DspB plasmid preps. We ran the PCR cleanup-kit on our DspB PCR product. We set up the digestion enzymes for Savinase. We will run the DspB and Savinase together on the low-melt gel when they are ready.

7/19/13

Neither the DspB or the Savinase showed up on the low-melts. We are re-doing the Savinase PCR using DNA from both the genomic purification kit and the PCR cleanup.

Savinase PCR -

Primers: 115 Forward, 116 Reverse

PCR Tubes:

Genomic Purification Kit
PCR Cleanup Kit
Control

7/22/13

We ran our PCR products from Friday (7/19) on gel. Columns 1 and 3 had nothing, but 2 had a strong Savinase band. Column 2 was our PCR cleanup kit sample. We then set up the restriction digests for both Savinase and DspB as outlined in the Restriction Digest Protocol in the Grose Lab Protocol Packet and using the following restriction enzymes and buffers:

Savinase - Hin III and Sac I with Buffer 2

DspB - EcoRI and XHO I with Buffer 4

7/26/13

We ran our E. coli transformations today on the Savinase and DspB restriction digest products using the Transformation protocol on the protocol page.

7/29/13

Our Savinase plate from the E. coli transformation has two colonies, however they didn’t look normal. The DspB didn’t have any colonies. We are re-running our ligations and E. coli transformations on these today. We also made new overnights and plated a new sample of cholera. The overnights and the new plate were all placed in the 30° incubator for ideal growth conditions.

7/30/13

We had colony growths for both Savinase and DspB. We did colony PCR using the Taq PCR reaction protocol listed on the protocol page.

7/31/13

We ran our PCR products on gel to check for the proper plasmids. We got products for Savinase clones colonies B & D as well as DspB. We set up LB-AMP overnights for Savinase clone colonies B & D and placed them in the shaker in the 37°C incubator.

We then set up restriction digests for DspB with the restriction enzymes EcoRI and XhoI. All procedures today followed protocols listed on the protocol page.