Team:Carnegie Mellon/Week2

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(Created page with "'''Monday, June 17th''' Oligos arrived<br> mRFP on plate 3 12N (BBa_E1010) <br> Top Row (left to right): ladder, KillerRed with BB promoters (SpeBBKRR and XbaBBKRF) x3, control ...")
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'''Monday, June 17th'''
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'''Monday June 10th:'''<br>
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Oligos arrived<br>
+
- Met with Natasa about modeling<br>
-
mRFP on plate 3 12N (BBa_E1010) <br>
+
- Made 0.5 L LB Agar and 0.5 L LB broth<br>
-
Top Row (left to right): ladder, KillerRed with BB promoters (SpeBBKRR and XbaBBKRF)  x3, control without DNA<br>
+
- Autoclaved<br>
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Bottom Row (left to right): RFP with EcoRFPfor and EcoRFPstR x3, control without DNA, ladder, RFP with SpeRBSRFPfor and PstSTRFPrev primers x3, control without DNA<br>
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- Added chloramphenicol to the agar<br>
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Purified PCR products (KR biobrick, RFP sequence)
+
- Poured plates (remember to put plates in fridge!)<br>
 +
- Looked through registry for Lac promoters<br>
 +
- Transformed 3 promoters (BBa_R0011, BBa_K864400, BBa_R0010) and killer red into E. coli<br>
 +
- BBa_R0010 and killer red on ampicillin, other two on chloramphenicol<br>
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Promoters<br>
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'''Tuesday, June 18'''<br>
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http://parts.igem.org/Part:BBa_R0011 plate 3, 5C (inconsistent) (chloramphenicol)
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Verified sequence of plasmid with ptac (H17) and WT lac (D1)<br>
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-lambda pL hybrid<br>
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Digest WT lac (D1), RFP, EGFP and KR with PstI and SpeI (2 hour digestion at 37ºC)<br>
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http://parts.igem.org/Part:BBa_K864400 plate 1, 17H (confirmed) (chloramphenicol)
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ends at 12:30pm<br>
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-ptac/trp/lac<br>
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Gel purify fragments in 1% agarose<br>
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http://parts.igem.org/Part:BBa_R0010 plate 5, 1D (confirmed) (Ampicillin)
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D1 - RFP - EGFP - KR - Ladder - Cheryl’s<br>
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-WT lac promotor<br>
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Media-preparing supplies arrived<br>
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Purified by GeneJet<br>
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Ligation by Thermo-Fisher  T4 ligase kit<br>
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6:2 µL RFP and EGFP ligations<br>
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2:2 µL KR since KR band was very strong<br>
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Transformation<br>
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2 min on ice<br>
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5 min at 42ºC<br>
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2 min  on ice<br>
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add 500 µL LB and incubate at 37ºC for 1 hr<br>
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Plate on LB/Amp plates and incubate overnight
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'''Wednesday, June 19'''<br>
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'''Tuesday June 11th:'''<br>
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RFP and KillerRed plates have many colonies EGFP has about 10 colonies.
+
Colonies on chloramphenicol plates had non-uniform size<br>
 +
possible that chloramphenicol was rather old<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>
 +
Ordered promoters and lambda arms/ packaging extract<br>
 +
We inoculated 4 cell cultures and stored them in 37 degree room<br>
 +
Cheryl is developing primers for cloning<br>
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Colony PCR for 4 EGFP, 4 KillerRed and 1 control of the plasmid DNA <br>
 
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Use VF2/VR primers<br>
 
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PCR ends at 4:50pm<br>
 
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Top Row (left to right): eGFP in pSC103 x4, KillerRed in psc103 plasmid x4, pSC103, ladder<br>
 
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Use gel to confirm the sequence<br>
 
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pSC103 is smaller than pSC103 with KR insert-transformation verified
 
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'''Thursday, June 20'''<br>
 
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KR cells cultured in tubes covered in aluminum foil<br>
 
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Observations:<br>
 
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Streaked plates grew successfully. Several colonies to choose from<br>
 
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KR cultures are very turbid (no color)<br>
 
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RFP cultures are bright red<br>
 
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EGFP culture is turbid (no color)<br>
 
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Negative control (VCS257) has no growth<br>
 
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Phage strains are all turbid<br>
 
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Digestion of EcoKR<br>
 
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Find tube of KR from 6/13<br>
 
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Digestion started: 10:40<br>
 
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Digestion started RFP 11:33<br>
 
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End digestion around 1:30<br>
 
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'''Friday, June 21'''<br>
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'''Wednesday, June 12'''<br>
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KR cells kept covered in aluminum foil until photobleaching <br>
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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>
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Diluted cells at 12:45pm in LB+amp<br>
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Mini-preps for all four plasmids<br>
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Note: amp should be added as 1/1000 dilution<br>
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Ran 2 uL DNA on gel- lanes right to left- standard; 5C lambda pL; 1D WT lac; 17H ptac/trp/lac; killer red
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Induction with IPTG: 1:45pm<br>
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Note: IPTG should be added as 1/500 dilution (6µL added to 3mL)<br>
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Check RFP for color change at 2:30 (no significant difference between induced and uninduced at 2:30 and 3pm)
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Photobleaching: 4-5pm?
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RFP + (light, 2 duplicates)<br>
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1. induced<br>
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2. not induced<br>
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RFP - (no light)<br>
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not induced
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KR + (light, 2 duplicates)<br>
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1. induced<br>
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2. not induced
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RFP - (no light)<br>
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not induced
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photobleaching measured with UV/Vis (where?) <br>
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6 LB/ amp plates at 10-5 dilution
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Inactivation of KillerRed is about 20 minutes according to Evrogen<br>
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http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml
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Inactivation of mRFP is about 10x faster than mCherry (which takes ~1.5-2 minutes)<br>
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http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf
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Inactivation of RFP takes >30 minutes at 100% light<br>
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Inactivation of KillerRed tubes were irradiated for 5 minutes at 100% light<br>
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All tube were plated (100µL) without dilution. KillerRed should have lower viable cell counts.
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�
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'''Saturday'''
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Rfp light, induced<br>
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Poor yield of 1D lac believed to be caused by pipetting error<br>
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Some uneven spreading but basically a lawn, fairly red
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Multiple bands in KillerRed lane probably because plasmid is large and was nicked during prep
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Rfp light, uninduced<br>
 
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More evenly spread, very small individual colonies observable near edges of plate. Both pink and non pink colonies
 
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Rfp no light uninduced<br>
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'''Thursday, June 13'''
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Some uneven spreading, lawn that is quite pink.
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PCR of KillerRed and Primers <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>
 +
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)
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Kr no light uninduced<br>
 
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Complete lawn, no color
 
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Kr light, induced<br>
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'''Friday, June 14'''<br>
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More or less a lawn (a few individual colonies observable), no observable color.<br>
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repeated PCR with KillerRed and primers SpeRBSKRF and PstStKRrev <br>
-
Kr light, uninduced<br>
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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>
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More uneven spreading than other two kr plates, some individual colonies observable, but it's basically a lawn. No color.
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made gel:
 +
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>
 +
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>
 +
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>
 +
iGEM1: ptac 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

Revision as of 20:57, 26 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

Retrieved from "http://2013.igem.org/Team:Carnegie_Mellon/Week2"