Team:Carnegie Mellon/Week2

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'''Monday June 10th:'''<br>
'''Monday June 10th:'''<br>
- Met with Natasa about modeling<br>
- Met with Natasa about modeling<br>
Line 19: Line 39:
'''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

Killer Red



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

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