Difference between revisions of "Team:Bio Without Borders/Notebook"
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| + | <a href="https://2018.igem.org/Team:Bio_Without_Borders/Protocols">Protocols</a> | ||
| + | <a href="https://2018.igem.org/Team:Bio_Without_Borders/LabBook">Lab Book</a> | ||
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Revision as of 18:26, 18 July 2018
Notebook
- Week 1 (June 4-8)
- •Made competent cells and aliquoted them into 25 1.5 mL tubes (50 microL each)
- Week 2 (June 11-15)
- •Testing competent cells using iGem DNA transformation samples.
- • 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 pSB1C3 using EcoR1 and Pst1
- •Made digest master mix; ran an ezyme digest of iGem plasmids pSB1A3 and pSB1K3 Performed ligation of
- pSB1A3 and pSB1K3; transformed pSB1A3 in competent cells 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 insert).
- •Streaked ampicillin (1A3) colony onto new plates.
- •Conducted cPCR for 1A3 colonies with 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.
- •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 j04450 insert with Q5 PCR to confirm size.
- •Amplification of pSB1C3 j04450 insert with Phusion PCR (because Q5 did not work). Confirmed size with gel electrophoresis.
- •Plasmid prep of pSB1A3
- •Digest of amplified pSB1C3 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) PSB1C3 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 (a.k.a. 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). 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.
- • Ran gel of digested backbone of BBa_E0040. (7/17)
- • Made plates of LB with chloramphenicol.
- • Performed transformation with 1uL 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).