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From 2014.igem.org

BYU 2014 Notebook Edit September October

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Week of September 5

Sept 3

(TR,CB) Performed SOEing PCR on the front and back halves of insert to go into pSR47s plasmid. We used the high fidelity q5 DNA polymerase.

Sept 4

(TR) Ran our PCR product from yesterday on a gel electrophoresis to determine if the SOEing worked. The gel came out with only a band at the 500bp range, therefore we know it didn't work. This shouldn't be a problem since we are still waiting to have some N. multiformis grow to be used in conjugation.

Week of September 12

Sept 8

(TR,CB) Ran SOEing PCR again with q5 as the polymerase.

Sept 10

(TR,CB) Ran a gel electrophoresis on Monday's PCR product. Still only a band at the 500bp mark. Reviewed literature on PCR techniques and tried to figure out why our SOEing isn't working. Didn't find anything new.

Sept 11

(TR) Checked on the growth of both the N. multiformis and N. eutropha. Neither one appears to have any growth. We cannot begin our work on N eutropha until be can grow enough to get a template from for PCR to make the front and back ends of our insert to knock out serB from the genome.

Week of September 19

Sept 15

(TR,CB) After collaboration, we decided to try purifying our PCR product of the front and back pieces of our eventual insert for ligation. We did this using the GFX PCR DNA and Gel Band Purification Kit. Then we ran the purified product on a gel at 120V for 30 min.

Sept 16

(TR) Took the purified product from yesterday and ran SOEing PCR again with q5 polymerase. After running it on a gel, we found that it worked! We also now have N multiformis growing which we obtained from the University of Utah, so we can move forward with conjugation as soon as our cloning is done and the N multiformis has grown up enough. We are abandoning the N eutropha possibility since we can't obtain any genomic DNA to use as a template.

Sept 17

(TR,CB) Took our SOEing PCR product and the vector and did a restriction digest of each with BamHI and SpeI using the standard protocol.

Sept 18

(TR,CB) We ran our digested insert and vector on a low melt gel at 80V for 60 min. After cutting out the correct bands, we went straight into a ligation. After the ligating we did a transformation into S17-1 E coli, plating onto LB kanamycin plates.

Sept 19

(TR) Came and checked our transformation, and found that only 1 colony grew. I decided to take the rest of the transformation that we saved in the fridge and didn't plate and plated it.

Week of September 26

Sept 20

(TR) Grabbed the plates out of the incubator from our transformation. One of the plates had 2 colonies. That isn't very much, but we will go ahead and check with colony PCR our 3 transformed colonies on Monday.

Sept 22

(TR,CB) Did our colony PCR using Taq polymerase and our vector primers and ran on a gel, after restreaking each of the colonies. The gel came out with only bands that are too small.

Sept 24

(TR,CB) Took our restreaked colonies and ran the colony PCR again with all the same conditions, except used our original SOEing primers instead of the vectors primers. Ran it on a gel and found that it still didn't work. We decided to try and redo everything from the ligation. So we redid the ligation with both a 1ul:5ul and 2ul:4ul vector to insert ratio. Then we transformed each one individually and plated all of the resuspended S17-1 onto its own plate.

Sept 25

(TR,CB) Looked at our plates and nothing grew. At this point we are too late to try and ligate again to finish conjugation by the competition, so we are abandoning this project until further notice and helping other groups on their projects.