Lethbridge/Notebook
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PCR Amplification of Riboswitch and CheZ | PCR Amplification of Riboswitch and CheZ | ||
| - | -made a 1/200 dilution of | + | -made a 1/200 dilution of maxiprep of pCheZ template |
-used spec to find concentration of 163ng/uL | -used spec to find concentration of 163ng/uL | ||
-made 1/10 and 1/100 dilutions of this for use as PCR template | -made 1/10 and 1/100 dilutions of this for use as PCR template | ||
Revision as of 20:25, 28 August 2007
Contents |
July 07 2007
Attempted Transformation of 4 Biobricks.
1. BBa_J5526 - Plate 3 well 6F - RFP 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 - GFP repressor
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. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
July 14 2007
RFP and GFP repressor plates had colonies (RFP had only 1 colony). GFP and promoter did not.
July 16 2007
Repeated transformation of GFP, RFP, and promoter, 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 and GFP repressor and inoculated amp+ LB broth
July 17 2007
GFP and promoter had colonies. RFP did not. Performed plasmid prep on RFP and GFP repressor 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 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
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 otherwise 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)
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