Ssbb14-Christopher J. Coates
~~!~~
Chris Notebook
List of all protocol used
http://openwetware.org/wiki/SBB10_protocols
Christopher J. Coates 21:11, 13 February 2014 (EST)
Group conversation established good candidate organisms. Settled on Thermus thermophilus and Thermatoga maldima, although Aquivex aeolicus was considered until logistics prevented acquisition of genomic DNA.
Feb 13. 2014.
Assigned Arginine-aaRS and Serine-aaRS from Thermatoga maritima.
Overall run down of Experiment:
Feb 27. 2014.
miniprep from cells pBad-*fill in this name* according to listed protocol.
Mar 4. 2014.
PCR according to construction files for ArgRS and SerRS synthesis.
Mar 6. 2014.
Ran analytical gel on Pcr pdt / ladder and previously isolated vector [ladder, trp, tyr, ArgRS, SerRS, [] [] [] vector] Zymo clean up of ArgRS and SerRS bands. Everything looked good and clean.
Mar 18. 2014.
Digested pcrpdt according to construction file using listed protocol.
Mar 20. 2014.
Ran prepatory gel ladder pBad pBad Arg Ser [] []. Everything looked clean but Ser band very very faint, re-perform digestion later.
Apr 1. 2014.
Digest vector Arg and Ser according to construction file and Re-ran prepatory gel, Banding came out clean, excised and stored.
Apr 3. 2014.
Zymo clean
Ligate
Transform
Plate
Was uncomfortable enough with procedure to perform sensitive/critical methods in one lab period (necessary) so waited until next lab. Others in lab group frequently failed to transform and ate up limited pcr product, having to re-perform the above in order to return to this step.
Apr 5. 2014.
Zymo clean digestion ArgRS and SerRS (according to protocol).
Ligate vect digest with ArgRS and SerRS according to construction file (according to protocol).
Transform ligation products into E.coli (according to heat shock protocol w/ recovery step)
Plate (according to protocol)
Apr 8. 2014.
Checked colonies, everything looked great (many well spaced colonies, no growth on negative control plate). Picked 2 Arg and 2 Ser now labelled Arg1, Arg2, Ser1, Ser2.
Inoculated picks into incubation growth media and informed Danny
Apr 10. 2014.
Miniprep incubated according to protocol. Determine which internal restrictions sites to use to restriction map. Settle on BamH1 for Arg and EcoR1 for Ser.
Apr 15. 2014.
Attempted to pour gel and digest, but was unable to get dams to make the gel. Had to remake gel after previous solidified. Will now always remember to find dams before microwaving gel. Not enough time to digest, run, and zymo clean samples so will leave new gel with 1x TAE in a pipette box for future use.
Apr 17. 2014.
Digest Arg products with BamH1 using listed protocol. Digest Ser products with EcoR1 using listed protocol. Run Gel Gel shows good banding for Ser, can send for sequencing. Ambiguous ghost banding for Arg, although it demonstrates the two expected digest bands on top of what was suggested to me to be uncut and monocut vector. Alternative mapping required. Miniprepped pBad
Apr 22. 2014.
Digest Arg and Vect with alternative restriction enzyme BglII Analysis suggests that Ser1 is correct and Arg1 is probably correct
May 1. 2014.
Run final gel to determine sequencing. Someone altered voltage to 180V and melted my gel mid run. No useful results. Submit samples based on previous evidence. Sequencing too late, never sent. However it seems clear from good colonies and clear early analytical gels that the part was transformed. And digestions partially confirm incorporation for Arg1 and definitely confirm for Ser1. Therefore the experiment has been reasonably carried.
There are a number of easy luciferase assays for ATP consumption available. It would have taken minimal resources to confirm activity of the t-RNA synthetases post boiling and this seems a logical next step. If these assays demonstrate little to no activity then a western blot may be performed to ensure expression of the fragment and stability after boiling. If expressing and non-functional then a different target should probably be chosen. Although ideally there would be enough final part targets to analyze multiple parts before abandoning the protein.
