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<dd>•Made competent cells and aliquoted them into 25 1.5 mL tubes (50 microL each)</dd> | <dd>•Made competent cells and aliquoted them into 25 1.5 mL tubes (50 microL each)</dd> | ||
<dt><b><font color="#009999">Week 2 (June 11-15)</font></b></dt> | <dt><b><font color="#009999">Week 2 (June 11-15)</font></b></dt> | ||
− | <dd>•Testing competent cells using iGem | + | <dd>•Testing competent cells using iGem pSB1C3.</dd> |
<dd>• Tested to see if they grew better on a freezer block versus an ice bath. They grow better in an ice bath.</dd> | <dd>• Tested to see if they grew better on a freezer block versus an ice bath. They grow better in an ice bath.</dd> | ||
<dd>• Ran colony PCR and plated bacteria on antimicrobial plate. </dd> | <dd>• Ran colony PCR and plated bacteria on antimicrobial plate. </dd> | ||
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<dt><b><font color="#009999">Week 3 (June 18-22)</font></b></dt> | <dt><b><font color="#009999">Week 3 (June 18-22)</font></b></dt> | ||
<dd>•Made chloramphenicol LB plates</dd> | <dd>•Made chloramphenicol LB plates</dd> | ||
− | <dd>•Restriction enzyme digest of iGem plasmid pSB1C3 using EcoR1 and Pst1</dd> | + | <dd>•Restriction enzyme digest of iGem plasmid backbone pSB1C3, pSB1A3, and pSB1K3 using EcoR1 and Pst1</dd> |
− | <dd> | + | <dd>•Ligated digested backbones with j04450 insert</dd> |
− | + | <dd>•Grew transformed competent cells with pSB1A3 with j04450 insert on ampicillin plate</dd> | |
− | <dd> | + | |
<dd>•Created a 50mg/mL kanamycin solution and then aliquoted it to about 80 1.5mL tubes. </dd> | <dd>•Created a 50mg/mL kanamycin solution and then aliquoted it to about 80 1.5mL tubes. </dd> | ||
− | <dd>•Inoculated LB with ampicillin colonies (with insert).</dd> | + | <dd>•Inoculated LB with ampicillin colonies (with j04450 insert).</dd> |
<dd>•Streaked ampicillin (1A3) colony onto new plates.</dd> | <dd>•Streaked ampicillin (1A3) colony onto new plates.</dd> | ||
− | <dd>•Conducted cPCR for | + | <dd>•Conducted cPCR for pSB1A3 colonies with j04450 insert.</dd> |
<dd>•Filled out first draft of safety form</dd> | <dd>•Filled out first draft of safety form</dd> | ||
<dd>•Went to Brighton Beach to examine horseshoe crabs' habitat and found a part of the carcass</dd> | <dd>•Went to Brighton Beach to examine horseshoe crabs' habitat and found a part of the carcass</dd> | ||
<dt><b><font color="#009999">Week 4 (June 25-29)</font></b></dt> | <dt><b><font color="#009999">Week 4 (June 25-29)</font></b></dt> | ||
− | <dd>•Conducted cPCR for pSB1K3.</dd> | + | <dd>•Conducted cPCR for pSB1K3 colonies with j04450 insert (the colonies took longer to turn red).</dd> |
<dd>•Ran 1% agarose gel of pSB1A3 with the insert (colonies that were re-streaked).</dd> | <dd>•Ran 1% agarose gel of pSB1A3 with the insert (colonies that were re-streaked).</dd> | ||
<dd>•Ran 1% agarose gel of pSB1K3 with insert.</dd> | <dd>•Ran 1% agarose gel of pSB1K3 with insert.</dd> | ||
<dd>•Cleaned laboratory material.</dd> | <dd>•Cleaned laboratory material.</dd> | ||
<dd>•Set up pSB1C3 backbone amplification PCR using Q5.</dd> | <dd>•Set up pSB1C3 backbone amplification PCR using Q5.</dd> | ||
− | <dd>•Ran gel of pSB1C3 | + | <dd>•Ran gel of pSB1C3 with Q5 PCR to confirm size.</dd> |
− | <dd>•Amplification of | + | <dd>•Amplification of j04450 insert with Phusion PCR (because Q5 did not work). Confirmed size with gel electrophoresis.</dd> |
<dd>•Plasmid prep of pSB1A3</dd> | <dd>•Plasmid prep of pSB1A3</dd> | ||
− | <dd>•Digest of amplified | + | <dd>•Digest of amplified j04450 insert (Phusion PCR).</dd> |
<dd>•Amplified linearized backbone from pSB1C3 and pSB1A3 with Phusion PCR</dd> | <dd>•Amplified linearized backbone from pSB1C3 and pSB1A3 with Phusion PCR</dd> | ||
<dd>•Ran a gel for pSB1A3 and pSB1C3 plasmid prep from day before to check band size</dd> | <dd>•Ran a gel for pSB1A3 and pSB1C3 plasmid prep from day before to check band size</dd> | ||
− | <dd>•Purified (accidentally-amplified with Phusion) | + | <dd>•Purified (accidentally-amplified with Phusion) j04450 insert, as well as the circular (made into linear) pSB1A3 and pSB1C3 backbones with PCR clean-up</dd> |
<dt><b><font color="#009999">Week 5 (July 2-9)</font></b></dt> | <dt><b><font color="#009999">Week 5 (July 2-9)</font></b></dt> | ||
<dd>•Went to Jones Beach to examine horseshoe crabs' habitat and found a carcass</dd> | <dd>•Went to Jones Beach to examine horseshoe crabs' habitat and found a carcass</dd> | ||
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<dd>• Inoculated LB broth with transformed cellulose binding domain containing colonies</dd> | <dd>• Inoculated LB broth with transformed cellulose binding domain containing colonies</dd> | ||
<dd>• Made new VF2 and VR primers and performed cPCR with them for BBa_K1478001 and pSB1K3</dd> | <dd>• Made new VF2 and VR primers and performed cPCR with them for BBa_K1478001 and pSB1K3</dd> | ||
− | <dd>• Ran gel for both pSB1K3 and BBa_K1478001 (failed again). Maybe we order new primers and change PCR master mix.</dd> | + | <dd>• 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.</dd> |
<dd>• Resuspended from distribution and transformed BBa_E1010(RFP,chloramphenicol backbone), BBa_K648013(GFP, chloramphenicol backbone) and BBa_E0040(GFP, ampicillin backbone) in competent cells.</dd> | <dd>• Resuspended from distribution and transformed BBa_E1010(RFP,chloramphenicol backbone), BBa_K648013(GFP, chloramphenicol backbone) and BBa_E0040(GFP, ampicillin backbone) in competent cells.</dd> | ||
<dd>• Plasmid prep of two pSB1K3 samples and two of cellulose binding domain BBa_K1478001 samples.</dd> | <dd>• Plasmid prep of two pSB1K3 samples and two of cellulose binding domain BBa_K1478001 samples.</dd> | ||
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<dd>• 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.</dd> | <dd>• 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.</dd> | ||
<dd>• cPCR was conducted for two BBa_E0040 (GFP) transformed colonies.</dd> | <dd>• cPCR was conducted for two BBa_E0040 (GFP) transformed colonies.</dd> | ||
− | <dd>• Ran gel of digested | + | <dd>•Digested E0040 (GFP) cPCR product (still contained pSB1A3 backbone). |
+ | <dd>• Ran gel of digested BBa_E0040 (a little shy of 720 bp). (7/17)</dd> | ||
<dd>• Made plates of LB with chloramphenicol.</dd> | <dd>• Made plates of LB with chloramphenicol.</dd> | ||
<dd>• 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).</dd> | <dd>• 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).</dd> | ||
− | <dd>• Did cPCR of | + | <dd>• Did cPCR of K648013 (GFP) and plasmid prep of GFP E0440.</dd> |
+ | <dd>• Ran digest of K648013 GFP</dd> | ||
Revision as of 20:54, 19 July 2018
Lab Book
- 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 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 (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; 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 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).
- • Did cPCR of K648013 (GFP) and plasmid prep of GFP E0440.
- • Ran digest of K648013 GFP