S-STEM Scholar Micheal Dockham's Blog

 This week has spent planning and prepping for the transformation of D. Ficus.  4.29 TGY media for plates was too concentrated agar wise, remade to 500ml from 250ml and re-autoclaved. 4.30 Poured 14 TGY plates for future inoculation.  2 TGY plates were inoculated w. D. Ficus cells were from February, may not grow. Need to observe.  Made 5x flask of 50ml of LB broth for future E. Coli inoculations.  .1ml/flask of amp(25mg/ml) 5.1 Inoculated 2x 20ml TGY flask with D. Ficus after positive gram stain results Inoculated 2x 50ml LB flask with E.Coli, added 100ul of 25mg/ml amp. Discussed further plans of plasmid extraction and component cell protocols and material request. Planned for transformation next week. 5.2     Gram stained all cultures, both E. coli and D. Ficus All good results Took OD values of chosen cultures.  E. Coli 2 OD 600 = 2.10 D. Ficus 2 OD 600 = 2.93 Held Biotech Club Meeting Prepped for Plasmid Extractions and Component Cell Protoco...

 This week has been spent in preperation for the ANAS conference that is taken place this weekend.  Weve spent the last week or so finishing up experiments, writing abstracts and drafting our poster that will be printed and displayed at the event.  I have mostly contributed to the formatting and designing of said poster, while the others have contributed greatly to the actual experiment. This has been great prep for the next and future conferences, as I will be presenting my own research and will have to go through this process again.

 This week was spent working productively on PCR reactions for both E. Coli and D. Aquaticus. The plan is to do a transformation with the cr2 gene, taken from the E.Coli using the pRAD1 vector in D. Aquaticus. We then ran gels, using electrophoresis to confirm the ~500bp and 7612bp sections METHOD  Multiple flask of TGY and LB+amp broth were made for our bacteria along with plates. Both were incubated at 37C for 24 hours. For E. Coli PCR, 50ul of culture was incubated at 95C for 10 minutes in an eppendorf tube. 4ul of culture were used and 2ul of Forward and Reverse Primers were added to the tube, along with 32ul of Master Mix. We then split this into 2x 20ul reactions for PCR. Thermocycler settings are shown in the photo below.  For our gel and confirmation of our 500bp cr2 gene, we ran a electrophoresis gel. This contained 2ul of PCR sample, 8ul of dH20 and 2ul dye. We then ran this with a 100bp ladder at 80Ma for roughly 15 minutes. See photo below For our D. Aquaticus...