Revision as of 14:54, 3 August 2018

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JUDGING FORM

Lab Book

This page documents all of our lab notes, featuring various protocols, experiments, and details about our wet and dry lab work that was conducted for our project.

Week 1 (June 4-8): •Made competent cells and aliquoted them into 25 1.5 mL tubes (50 µl each)
Week 2 (June 11-15): •Testing competent cells using iGem pSB1C3.; • Tested to see if they grew better on a freezer block versus an ice bath. They grow better in an ice bath.; • Ran colony PCR and plated bacteria on antimicrobial plate.; • cPCR, chloramphenicol, and culturing protocols; • Ran 1% agarose gel for cPCR; • Plasmid prep of pSB1C3
Week 3 (June 18-22): •Made chloramphenicol LB plates; •Restriction enzyme digest of iGem plasmid backbone pSB1C3, pSB1A3, and pSB1K3 using EcoR1 and Pst1; •Ligated digested backbones with j04450 insert; •Grew transformed competent cells with pSB1A3 with j04450 insert on ampicillin plate; •Created a 50mg/mL kanamycin solution and then aliquoted it to about 80 1.5mL tubes.; •Inoculated LB with ampicillin colonies (with j04450 insert).; •Streaked ampicillin (1A3) colony onto new plates.; •Conducted cPCR for pSB1A3 colonies with j04450 insert.; •Filled out first draft of safety form; •Went to Brighton Beach to examine horseshoe crabs' habitat and found a part of the carcass
Week 4 (June 25-29): •Conducted cPCR for pSB1K3 colonies with j04450 insert (the colonies took longer to turn red).; •Ran 1% agarose gel of pSB1A3 with the insert (colonies that were re-streaked).; •Ran 1% agarose gel of pSB1K3 with insert.; •Cleaned laboratory material.; •Set up pSB1C3 backbone amplification PCR using Q5.; •Ran gel of pSB1C3 with Q5 PCR to confirm size.; •Amplification of j04450 insert with Phusion PCR (because Q5 did not work). Confirmed size with gel electrophoresis.; •Plasmid prep of pSB1A3; •Digest of amplified j04450 insert (Phusion PCR).; •Amplified linearized backbone from pSB1C3 and pSB1A3 with Phusion PCR; •Ran a gel for pSB1A3 and pSB1C3 plasmid prep from day before to check band size; •Purified (accidentally-amplified with Phusion) j04450 insert, as well as the circular (made into linear) pSB1A3 and pSB1C3 backbones with PCR clean-up
Week 5 (July 2-9): •Went to Jones Beach to examine horseshoe crabs' habitat and found a carcass; •Worked on GoFundMe campaign page; •Digested pSB1K3 backbone, ligated it with the J04450 insert (from purified J04450 insert from 6/27), and transformed cells for Kanamycin resistance & plated them; •Ran cPCR for transformed kanamycin resistant colonies; •Coded format for iGEM website; •Inoculated LB broth with transformed kanamycin resistant colonies; •Made iGEM Facebook page
Week 6 (July 7-13): •Human practices: PCR and Pizza.; •Ran cPCR of pSB1K3 ith j04450 insert.; •Re-did cPCR of pSB1K3 and ran gel (failed again).; •Made chloramphenicol solution, as well as chloramphenicol plates.; •Transformed the cellulose binding domain (BBa_K1478001) from distribution plate into competent cells and plated them on chloramphenicol.; (We need to figure out which primers to so that we can observe a band from cPCR of pSB1K3).; • Inoculated LB broth with transformed cellulose binding domain containing colonies; • Made new VF2 and VR primers and performed cPCR with them for BBa_K1478001 and pSB1K3; • Ran gel for both pSB1K3 and BBa_K1478001 (failed again; a band seemed to form at ~700bp for K1478001), but is not the correct length of 309bp). Maybe we order new primers and change PCR master mix.; • Resuspended from distribution and transformed BBa_E1010(RFP,chloramphenicol backbone), BBa_K648013(GFP, chloramphenicol backbone) and BBa_E0040(GFP, ampicillin backbone) in competent cells.; • Plasmid prep of two pSB1K3 samples and two of cellulose binding domain BBa_K1478001 samples.; • Plated all transformation cells
Week 7 (July 16-20): • BBa_E1010(RFP,chloramphenicol backbone) and BBa_K648013(GFP, chloramphenicol backbone) transformed plates did not work. BBa_E0040(GFP, ampicillin backbone) transformed plate had two colonies present.; • cPCR was conducted for two BBa_E0040 (GFP) transformed colonies.; •Digested E0040 (GFP) cPCR product (still contained pSB1A3 backbone).; • Ran gel of digested BBa_E0040 (a little shy of 720 bp). (7/17); • Made plates of LB with chloramphenicol.; • Performed transformation with 1 µl of E1010(RFP) and K648013(GFP), on chloramphenicol. Result: There were no colonies on plates with E1010(RFP),and approximately 3 or 4 colonies grew on plate with K648013(GFP).; • Did cPCR of K648013 (GFP) and plasmid prep of GFP E0440.; • Ran digest of K648013 GFP (unecessary because we accidentally used VF2 and VR during cPCR which only amplified the insert, rather than the backbone, which makes the digest uncessary) and confirmed size with gel electrophoresis; • Did plasmid prep of GFP k648013.; • Performed transformation of k1463560 (CBD) and K648013 (GFP) and plated them. Also inoculated the left over k14 and k648013 with 200 µl of SOC and incubated.
Week 8 (July 23-27): • Performed protocols for the Interlab study: Used DH5-Alpha strain of bacteria to perform a transformation on 8 different plates.; • The next day we observed colonies in each of the plates, so we inoculated two colonies from each plate in 5mL of LB+Chloramphenicol and put them on the rotator to grow overnight.; • Attempted to work with Fluostar Galaxy for interlab measurement study. Veered off course and put cultures in the fridge. Used Genesys 20 to measure fluorescence and absorbance of cultures.; • Contacted FLUOstar technician to resolve problems with machine and redid measurements of cultures. Conducted all cell calibration studies for Interlab and recorded data.; • Measured absorbance and fluorescence for Hour 0 and Hour 6 plates with cell cultures.
Week 9 (July 30-August 3): • Started redoing the measurement study. Inoculated colonies in LB+chloramphenicol.; • Conducted calibration studies with FLUOstar Galaxy.; • Measured absorbance and fluorescence for Hour 0 and Hour 6 plates with cell cultures.; • Used Genesys 20 to measure 1:10 dilutions to bring absorbance to .1

@@ Line 84: / Line 84: @@
 <dd>• Performed protocols for the Interlab study: Used DH5-Alpha strain of bacteria to perform a transformation on 8 different plates.</dd>
 <dd>• The next day we observed colonies in each of the plates, so we inoculated two colonies from each plate in 5mL of LB+Chloramphenicol and put them on the rotator to grow overnight.</dd>
-<dd>• Attempted to work with Fluostar Galaxy for interlab measurement study. Veered off course and put cultures in the fridge. Used Thermo Genesys 20 to measure fluorescence and absorbance of cultures.</dd>
+<dd>• Attempted to work with Fluostar Galaxy for interlab measurement study. Veered off course and put cultures in the fridge. Used Genesys 20 to measure fluorescence and absorbance of cultures.</dd>
 <dd>• Contacted FLUOstar technician to resolve problems with machine and redid measurements of cultures. Conducted all cell calibration studies for Interlab and recorded data.</dd>
 <dd>• Measured absorbance and fluorescence for Hour 0 and Hour 6 plates with cell cultures.</dd>
@@ Line 93: / Line 93: @@
 <dd>• Conducted calibration studies with FLUOstar Galaxy.</dd>
 <dd>• Measured absorbance and fluorescence for Hour 0 and Hour 6 plates with cell cultures.</dd>
-<dd>• Used Thermo Genesys 20 to measure 1:10 dilutions to bring absorbance to .1</dd>
+<dd>• Used Genesys 20 to measure 1:10 dilutions to bring absorbance to .1</dd>

Difference between revisions of "Team:Bio Without Borders/LabBook"

Revision as of 14:54, 3 August 2018

Lab Book