Lethbridge/Notebook
From 2007.igem.org
Contents |
July 07 2007
Attempted Transformation of 4 Biobricks.
1. BBa_J5526 - Plate 3 well 6F - RFP comp. 2. BBa_I13522 - Plate 2 well 15H - GFP 3. BBa_R0011 - Plate 1 well 7M - LacI inhibited promoter 4. BBa_PO440 - Plate 1 well 21K - tet R
Protocol
1. Add 15uL deionized H2O to IGEM plate well 2. Add 1 uL plasmid to 25uL DH5alpha 3. Incubate on ice for 30min 4. Heat shock without shaking for 20sec 5. Place on ice for 2min 6. Add 0.5mL LB, incubate for 1hr 7. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
July 14 2007
RFP and tetR had colonies (RFP comp. had only 1 colony). GFP and Lac pr. did not.
July 16 2007
Repeated transformation of GFP, RFP comp., and Lac pr., with addition of a cell concentraion step and more cells/plasmid. (Revisions to protocol in bold.)
Protocol
1. Add 35uL deionized H2O to well 2. Add 5uL plasmid to 50uL DH5alpha 3. Incubate on ice for 30min 4. Heat shock without shaking for 20sec 5. Place on ice for 2min 6. Add 0.5mL LB, incubate for 1hr 7. Spin down cells gently, remove 350uL supernatant and resuspend (gently!) pellet 8. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
Picked colonies from RFP comp. and tetR and inoculated amp+ LB broth
July 17 2007
GFP and Lac pr. had colonies. RFP comp. did not. Performed plasmid prep on RFP and tetR cultures according to QIAprep Miniprep Handbook 2nd Ed. Nov '05 rip out bench protocol for microcentrifuge.
July 18 2007
Ran extracted plasmids on a gel
RFP comp. appears to be too small, and cells do not show red flourescence under UV light. Since part appears not to be working, will build RFP part from 5 subparts.
July 23 2007
Attempted transformation of 6 biobricks
1. BBa_I13522 - plate 2 well 15H - GFP
-had to redo GFP because accidentally transformed plate 1 well 15H the first time
2. BBa_C0012 - plate 1 well 5A - Lac I
3. BBa_B0034 - plate 1 well 3O - RBS
4. BBa_E1010 - plate 2 well 15M - RFP basic
5. BBa_B0010 - plate 2 well 3P - T1
6. BBa_B0012 - plate 1 well 1C - T2
Protocol
1. Add 15uL deionized H2O to well 2. Add 1 uL plasmid to 25uL DH5alpha 3. Incubate on ice for 30min 4. Heat shock without shaking for 20sec 5. Place on ice for 2min 6. Add 0.5mL LB, incubate for 1hr 7. Spin 13000 rpm for 1min, remove 400uL supernatant and resuspend 7. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
July 24 2007
Note book created (All previous entries are transcribed from paper lab notebook)
All transformations worked except LacI. Picked two colonies from each and cultured in 5mL amp+ LB, incubated overnight at 37 C.
August 14 2007
PCR Amplification of Riboswitch and CheZ
-made a 1/200 dilution of maxiprep of pCheZ template -used spec to find concentration of 163ng/uL -made 1/10 and 1/100 dilutions of this for use as PCR template
Riboswitch PCR (recipe/reaction)
5uL 5x phusion polymerase buffer 1uL dNTP mix 1.25uL 20uM Forward Primer 1.25uL 20uM Reverse Primer 0.5uL Phusion polymerase 11.5uL H2O (optima) 5uL template (1/10, 1/100, no DNA control)
CheZ PCR (recipe/reaction)
5uL 5x phusion polymerase buffer 1uL dNTP mix 1.25uL 20uM Forward Primer 1.25uL 20uM Reverse Primer 0.5uL Phusion polymerase 11.5uL H2O (optima) 5uL template (1/10, 1/100, no DNA control)
Reaction Conditions
1X 98 C 1.5min
35X 98 C 30sec
58 C 45sec
72 C 2.5min
1X 72 C 4min
Ran on 0.8% agarose gel with HindIII marker
PCR did not work correctly, witnessed only primer dimer and plasmid bands
Will redo with a much shorter extension time as CheZ is only 640bp
Will redo with a better size marker and a higher percentage agarose gel
August 20 2007
PCR Amplification of Riboswitch and CheZ
Used same template as August 14
CheZ PCR (recipe/reaction)
5uL 5x phusion polymerase buffer 1uL dNTP mix 1.25uL 20uM Forward Primer 1.25uL 20uM Reverse Primer 0.5uL Phusion polymerase 11.5uL H2O (optima) 5uL template (1/10, 1/100, no DNA control)
New reaction conditions
1X 98 C 1min
35X 98 C 15sec
60 C 20sec
72 C 30min
1X 72 C 4min
1.5% Agarose gel
noticed contamination in no DNA control, will re-make primers, replace H2O stocks and re-do. band appeared in the correct spot
August 22 2007
Re-made primers from stock solution
Will modify PCR reaction conditions to take into account the low Tm of the regions that actually bind, and then raise annealing temperature after a few cycles when the primer binding regions has been amplified
Modified Reaction Conditions
1X 98 C 1min
5X 98 C 20sec
41 C 30sec
72 C 1min
30X 98 C 20sec
60 C 30sec
72 C 1min
1X 72 C 4min
New PCR recipie (only minor changes)
5uL 5x phusion polymerase buffer 1uL dNTP mix 1uL 20uM Forward Primer 1uL 20uM Reverse Primer 0.25uL Phusion polymerase 10.75uL H2O (optima) 1uL template (1/10, 1/100, no DNA control)
August 27 2007
Attempted Transformation of 2 Biobricks using XL1-blue supercompotent cells.
1. BBa_I13507 - Plate 1 well 16N - RFP sub. 2. BBa_C0012 - Plate 1 well 5A - LacI (repeat)
Protocol
1. Add 50uL XL1-blue cells with 0.85uL 2-mercaptoethanol to 15mL falcon tube (prechilled) 2. Leave on ice for 10 min. 3. Add 15uL deionized H2O to IGEM plate well 4. Add 1 uL plasmid to cells 5. Incubate on ice for 30min 6. Heat shock without shaking for 45sec at 42 C. 7. Place on ice for 2min 8. Add 0.9mL SOC media, preheated to 42 C, incubate for 1hr 9. Plate 200uL on amp+ LB plates, incubate overnight at 37 C
Ran a digest of Lac pr. and tetR plasmids with the intention of ligating tetR behind Lac pr.
reaction conditions 1. 2uL NEB 2 stock buffer 2. 0.2uL stock BSA 3. 5uL prepped plasmid 4. 0.5uL of each restriction enzyme (two) 5. 11.8uL dH2O Used Pst1 + Spe1 for Lac. pr. and used Pst1 + Xba1 for tetR.
August 28 2007
RFP sub. transformation worked, but LacI did not. Picked colonies from RFP sub. and incubated in amp/LB overnight at 37 C. Supervisor informed that supercompotent cells used had been in a thaw accident, repeated LacI and RFP comp. transformations with new supercompotent cells using the same protocol
August 29 2007
RFP comp. worked (1 colony) but LacI once again did not. picked RFP comp. incubated in amp/LB overnight at 37 C.
Since LacI is not working, we are switching the repressor component of the reporter system. Lambda cI and a lambda cI inhibited promoter will be used instead of the LacI components.
Attempted Transformation of 3 Biobricks using XL1-blue supercompotent cells with same protocol as 27/08/07.
1. BBa_R0051 - Plate 1 well 9C - lambda cI inhibited promoter 2. BBa_C0051 - Plate 1 well 5G - lambda cI 3. BBa_J23119 - PLate 3 well 19A - Strong promoter for riboswitch
August 30 2007
All three transformations worked, picked two colonies from each and incubated overnight at 37 C in LB/amp
The RFP comp. colony picked on the 29th did not grow, but the plates had been left in teh incubator for another night and colonies grew on both the LacI and RFP comp. plate. Probably false positives, but picked two colonies from each and inoculated LB/amp
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September 3 2007
Created a new streak plate of RP1616 (cheZ mutant) to use with Fermentas TransformAid Kit to make cells competant for transformation
September 5 2007
Performed pruification of the PCR product of CheZ using Qiagen MinElute Kit for PCR purification.
Started overnight culture of RP1616 to use for transformation of Promoter+Riboswitch+CheZ plasmid as well as a puc19 control.
