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Team:Carnegie Mellon/Week2
From 2013.igem.org
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'''Tuesday June 11th:'''<br> | '''Tuesday June 11th:'''<br> | ||
| - | Colonies on chloramphenicol plates had non-uniform size<br> | + | -Colonies on chloramphenicol plates had non-uniform size<br> |
| - | possible that chloramphenicol was rather old<br> | + | ---possible that chloramphenicol was rather old<br> |
| - | Left LB/cam plates out all night :(<br> | + | -Left LB/cam plates out all night :(<br> |
| - | plated with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 to see if plates are still ok<br> | + | ---plated with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 to see if plates are still ok<br> |
| - | Ordered promoters and lambda arms/ packaging extract<br> | + | -Ordered promoters and lambda arms/ packaging extract<br> |
| - | We inoculated 4 cell cultures and stored them in 37 degree room<br> | + | -We inoculated 4 cell cultures and stored them in 37 degree room<br> |
| - | Cheryl is developing primers for cloning<br> | + | -Cheryl is developing primers for cloning<br> |
'''Wednesday, June 12'''<br> | '''Wednesday, June 12'''<br> | ||
| - | Plates with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 confirmed that plates are ok. Plate with strain that was only amp resistant had no colonies, two plates with chloramphenicol resistant plasmids had individual colonies, not a bacterial lawn (yay!)<br> | + | -Plates with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 confirmed that plates are ok. Plate with strain that was only amp resistant had no colonies, two plates with chloramphenicol resistant plasmids had individual colonies, not a bacterial lawn (yay!)<br> |
| - | Mini-preps for all four plasmids<br> | + | -Mini-preps for all four plasmids<br> |
| - | Ran 2 uL DNA on gel- lanes right to left- standard; 5C lambda pL; 1D WT lac; 17H ptac/trp/lac; killer red | + | ---Ran 2 uL DNA on gel- lanes right to left- standard; 5C lambda pL; 1D WT lac; 17H ptac/trp/lac; killer red |
| - | Poor yield of 1D lac believed to be caused by pipetting error<br> | + | ---Poor yield of 1D lac believed to be caused by pipetting error<br> |
| - | Multiple bands in KillerRed lane probably because plasmid is large and was nicked during prep | + | ---Multiple bands in KillerRed lane probably because plasmid is large and was nicked during prep |
'''Thursday, June 13''' | '''Thursday, June 13''' | ||
| - | PCR of KillerRed and Primers <br> | + | -PCR of KillerRed and Primers <br> |
| - | Gel of PCR products: <br> | + | --Gel of PCR products: <br> |
Top row (left to right): ladder, Killer Red x3 with EcoKRF and EcoKRstR primers, eGFP x3 with EcoEGFPfor and EcoEGFPstR primers, Killer Red x3 with SpeRBSKRF and PstStKRrev primers<br> | Top row (left to right): ladder, Killer Red x3 with EcoKRF and EcoKRstR primers, eGFP x3 with EcoEGFPfor and EcoEGFPstR primers, Killer Red x3 with SpeRBSKRF and PstStKRrev primers<br> | ||
| - | Bottom row (left to right): first two are Cheryls, no DNA controls x4 (For: KillerRed1, eGFP, KillerRed2, promoters from left to right), pTac/trp/lac, WT lac, lambda pl, ladder (SpeRBSKRF, PstStKRrev primers for promoters) | + | Bottom row (left to right): first two are Cheryls, no DNA controls x4 (For: KillerRed1, eGFP, KillerRed2, promoters from left to right), pTac/trp/lac, WT lac, lambda pl, ladder (SpeRBSKRF, PstStKRrev primers for promoters) <br> |
| - | + | <html><img src="https://static.igem.org/mediawiki/2013/4/41/CMUGel1.png"</html> | |
'''Friday, June 14'''<br> | '''Friday, June 14'''<br> | ||
| - | repeated PCR with KillerRed and primers SpeRBSKRF and PstStKRrev <br> | + | -repeated PCR with KillerRed and primers SpeRBSKRF and PstStKRrev <br> |
| - | primers were long and were at room temp for several minutes before cycler started, so we think they formed primer dimers based on gel from June 13<br> | + | ---primers were long and were at room temp for several minutes before cycler started, so we think they formed primer dimers based on gel from June 13<br> |
| - | made gel: | + | -made gel: |
| - | 0.4g agarose, 40ml TE buffer, swirl to break up agarose. microwave 1 min 30s in erlenmeyer<br> | + | ---0.4g agarose, 40ml TE buffer, swirl to break up agarose. microwave 1 min 30s in erlenmeyer<br> |
| - | Swirl again to prevent agarose settling and add 2 \micro\ liters ethidium bromide and swirl to mix<br> | + | ---Swirl again to prevent agarose settling and add 2 \micro\ liters ethidium bromide and swirl to mix<br> |
| - | Pour gel in taped mold<br> | + | ---Pour gel in taped mold<br> |
| - | Decided to clone KillerRed into plasmid (high copy?) first because phage parts won’t be here until next Wednesday<br> | + | -Decided to clone KillerRed into plasmid (high copy?) first because phage parts won’t be here until next Wednesday<br> |
| - | Sent ptac promoter DNA (H17) and WT EColi lac promoter to be sequenced (s/b here by tues)<br> | + | -Sent ptac promoter DNA (H17) and WT EColi lac promoter to be sequenced (s/b here by tues)<br> |
| - | 5 μL H_2O, 5 μL DNA, 1 μL 10 μM primer (VF2)<br> | + | ---5 μL H_2O, 5 μL DNA, 1 μL 10 μM primer (VF2)<br> |
| - | iGEM1: ptac promoter<br> | + | ---iGEM1: ptac promoter<br> |
| - | iGEM2: WT E. Coli lac promoter<br> | + | ---iGEM2: WT E. Coli lac promoter<br> |
| - | From left to right: ladder, KillerRed template with primers SpeRBSKRF and PstStKRrev x2, control and KillerRed with primers as first 2 rows (whoops) and control | + | -From left to right: ladder, KillerRed template with primers SpeRBSKRF and PstStKRrev x2, control and KillerRed with primers as first 2 rows (whoops) and control <br> |
| + | <html><img src="https://static.igem.org/mediawiki/2013/6/67/Cmugel2.png"></html> | ||
Latest revision as of 23:22, 27 September 2013
Monday June 10th:
- Met with Natasa about modeling
- Made 0.5 L LB Agar and 0.5 L LB broth
- Autoclaved
- Added chloramphenicol to the agar
- Poured plates (remember to put plates in fridge!)
- Looked through registry for Lac promoters
- Transformed 3 promoters (BBa_R0011, BBa_K864400, BBa_R0010) and killer red into E. coli
- BBa_R0010 and killer red on ampicillin, other two on chloramphenicol
Promoters
http://parts.igem.org/Part:BBa_R0011 plate 3, 5C (inconsistent) (chloramphenicol)
-lambda pL hybrid
http://parts.igem.org/Part:BBa_K864400 plate 1, 17H (confirmed) (chloramphenicol)
-ptac/trp/lac
http://parts.igem.org/Part:BBa_R0010 plate 5, 1D (confirmed) (Ampicillin)
-WT lac promotor
Tuesday June 11th:
-Colonies on chloramphenicol plates had non-uniform size
---possible that chloramphenicol was rather old
-Left LB/cam plates out all night :(
---plated with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 to see if plates are still ok
-Ordered promoters and lambda arms/ packaging extract
-We inoculated 4 cell cultures and stored them in 37 degree room
-Cheryl is developing primers for cloning
Wednesday, June 12
-Plates with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 confirmed that plates are ok. Plate with strain that was only amp resistant had no colonies, two plates with chloramphenicol resistant plasmids had individual colonies, not a bacterial lawn (yay!)
-Mini-preps for all four plasmids
---Ran 2 uL DNA on gel- lanes right to left- standard; 5C lambda pL; 1D WT lac; 17H ptac/trp/lac; killer red
---Poor yield of 1D lac believed to be caused by pipetting error
---Multiple bands in KillerRed lane probably because plasmid is large and was nicked during prep
Thursday, June 13
-PCR of KillerRed and Primers
--Gel of PCR products:
Top row (left to right): ladder, Killer Red x3 with EcoKRF and EcoKRstR primers, eGFP x3 with EcoEGFPfor and EcoEGFPstR primers, Killer Red x3 with SpeRBSKRF and PstStKRrev primers
Bottom row (left to right): first two are Cheryls, no DNA controls x4 (For: KillerRed1, eGFP, KillerRed2, promoters from left to right), pTac/trp/lac, WT lac, lambda pl, ladder (SpeRBSKRF, PstStKRrev primers for promoters)
Friday, June 14
-repeated PCR with KillerRed and primers SpeRBSKRF and PstStKRrev
---primers were long and were at room temp for several minutes before cycler started, so we think they formed primer dimers based on gel from June 13
-made gel:
---0.4g agarose, 40ml TE buffer, swirl to break up agarose. microwave 1 min 30s in erlenmeyer
---Swirl again to prevent agarose settling and add 2 \micro\ liters ethidium bromide and swirl to mix
---Pour gel in taped mold
-Decided to clone KillerRed into plasmid (high copy?) first because phage parts won’t be here until next Wednesday
-Sent ptac promoter DNA (H17) and WT EColi lac promoter to be sequenced (s/b here by tues)
---5 μL H_2O, 5 μL DNA, 1 μL 10 μM primer (VF2)
---iGEM1: ptac promoter
---iGEM2: WT E. Coli lac promoter
-From left to right: ladder, KillerRed template with primers SpeRBSKRF and PstStKRrev x2, control and KillerRed with primers as first 2 rows (whoops) and control
