Team:Lethbridge/Notebook
From 2009.igem.org
(→September 4=) |
(→Sept 10) |
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Line 5,587: | Line 5,587: | ||
#C-term-dT + C-EYFP #2 | #C-term-dT + C-EYFP #2 | ||
#pSB1A3-1+N-CFP#2 | #pSB1A3-1+N-CFP#2 | ||
+ | #pSB1A3-1+N-ECFP#2 | ||
#lumazine+sRBS | #lumazine+sRBS | ||
#pSB1A3-2+C-EYFP#1 | #pSB1A3-2+C-EYFP#1 | ||
+ | #pSB1A3-1+N-EYFP#2 | ||
#mRBS-N-term +N-CFP#1 | #mRBS-N-term +N-CFP#1 | ||
#mmS6+pSB1A3-1 | #mmS6+pSB1A3-1 |
Revision as of 05:14, 18 September 2009
Calendar
MayMay 5Roxanne, Megan, Alix, Mackenzie, Sebastian
May 6Roxanne
May 12Roxanne
Roxanne, Alix, Ashley, Megan, Mackenzie, Kirsten
May 13Roxanne
May 14Roxanne, Alix, Ashley, Mackenzie, Kirsten
May 19Roxanne, Alix, Ashley, Kirsten, Megan, Mackenzie
Lane 1: Ladder Lane 2: ohbR1 Lane 3: ohbR2 Lane 4: ohbC Lane 5: ohbB1 Lane 6: ohbB2 Lane 7:ohbA Lane 8:ohbC Lane 9: ohbB1 Lane 10:ohbB2
May 21Roxanne
May 26Roxanne, Alix, Megan, Mackenzie, Kirsten
May 28Roxanne
Roxanne, Ashley, Alix, Kirsten, Mackenzie, Megan
Roxanne
May 29Roxanne
JuneJune 1Roxanne
Lane 1: ladder Lane 2: ohbA (conc.1x) Lane 3: ohbA (conc.1/10) Lane 4: ohbB(1x) Lane 5: ohbB(1/10) Lane 6: ohbC(1x) Lane 7: ohbC(1/10) Lane 8: ohbR (1x) Lane 9: ohbR(1/10) Lane 10: ohbR(1x)
Mackenzie & Roxanne
June 3Roxanne
June 4
June 15Transformed the pTet and EYFP genes from the Biobrick registry
June 16
June 17
June 18
in order to send for sequencing with the UR and UF2 primers June 22
June 23
Lane 1: 1kb ladder Lane 2:EYFP1A Lane 3: EYFP1B Lane 4: EYFP1B runover Lane 5: EYFP2A Lane 6: EYFP2B Lane 7: pTet-1A Lane 8: pTet-1B Lane 9: pTet2-A Lane 10: pTet-2B
June 24
June 25
Lane 1: 1kb ladder Lane 2: Control-no enzyme(pTet) Lane 3: pTet-pstI Lane 4: pTet-SpeI Lane 5: pTet-SpeI/PstI Lane 6:EYFP-no enzyme Lane 7: EYFP-PstI Lane 8: EYFP-XbaI Lane 9: EYFP-XbaI/PstI Lane 10: 1kb ladder
June 26
June 30
JulyJuly 2
July 3
Lane 2: 10uL of 2kb ladder, Lane 4:undiluted pTet Lane 5: undiluted CFP Lane 6:undiluted EYFP Lane 8: 1/10pTet Lane 9: 1/10 CFP Lane 10: 1/10 EYFP Lane 12: 1/100 pTet Lane 13: 1/100 CFP Lane 14: 1/100 EYFP Lane 16: 1/1000pTet Lane 17: 1/1000 CFP Lane 18: 1/1000 EYFP July 6thJeff, Mackenzie, Ashley Using 1/10 and 1/100 dilutions of pTET and EYFP to perform large scale digests.
4 reactions each:
100µL each, means we need 400µL total for each. 1/10 dilution=40µL DNA, 360µL water 1/100 dilution=4µL DNA, 396µL water Reaction set up:
16 reactions run overnight @ 37° C Kirsten, Lisza TetR Q04400-plate 1 well 16p-pSB2K3 LacI promoter-R0010-plate 1 well 1d-Amp pBAD-I13453-plate 1, well 1n-pSB1A2 strong RBS-B0030-plate 1 well 1h-pSB1A2 med RBS-B0030-plate 1 well 2I-pSB1A2 10µL water into wells Transformation: 2µL DNA (TetR, LacI, pBAD, med RBS, strong RBS) into 25µL competent DH5α cells (from the -80 fridge). Pipet up then down and swirl. One ice for 30min, then heat shock at 42° C for 45 sec. Ice for 1 min, add 250µL LB broth (from fridge) and incubate in shaker for 1 hour. Plate onto ampicillin plates (pBAD, LacI, strond and med RBS) and kanamycin plate (TetR) into incubator overnight. Control: 2.5µL water and 2.5µL DH5α.
July 8Jeff Overnight cultures (200mL + antibiotics) of:
Centrifuged at 5000g, 10 min, 4° for maxiprep 500µL each of each culture used to make glycerol stocks – duplicates made, stored in -80° freezer after flash freezing with liquid nitrogen. EYFP insert from AGE separation (incubated in 600µL buffer QG overnight):
Flow through replaced in original tube. 500µL QG applied to column, 1 min centrifugation. Colum washed w/ 750µL PE buffer (1 min centrifuge). Flow through discarded. 1 more min of centrifugation to get rid of buffer PE. DNA eluted w/ 25µL water (37°C for 10 min) also a second water elution w/ 25µL water (E2). 1/10 double digest of pTet heat incubated at 85°C for 10 min. Maxipreps: 3mL ALSI added, cells responded. 500µL lysozyme added. 15 min @RT, 6mL ALSII added. 10 min at RT. 4.5mL cold ALSIII added, 10 min on ice. Left a 4°C July 13Setting up ligations: 1µL ligase (stock) 10x T4 DNA ligase buffer 1/10 double digested pTet est 20ng/µL E1 (60% yield) = ~5ng/µL; ~1/3 insert size/pTet Control needed:
Reaction 1:
Reaction 2:
Reaction 3:
Reaction 4:
Incubated at 37°C overnight Kirsten, Mackenzie Chloroform extraction of medRBS, pLacI, TetR, that Jeff started. pBab & strong RBS cloudy and phenol chloro done twice.
July 14Megan, Mackenzie, Alix Transformations of ligation reactions Took 2µL of DNA from 1, 2, 3, 4 and added to 25 µL of DH5α cells. Incubated on ice for 30 min. Heat shocked at 42°C for 45 sec. Back to ice for 1 min. For ligations add 500µL of LB broth to each tube, working sterile using a Bunsen burner. Incubate in shaker at 37°C for1 hour at 200 rpm. Made up two plates (Amp) of each, 100µL and 400µL. July 16thAshley, Mackenzie, Roxanne Pelleted pTet-EYFP ligation reactions 3 and 4 using at 13000rpm for 1 min. Mini-prep of pTet-EYFP ligation reactions 3 and 4 using Qiagen kit + protocol. July 20thLisza Our genes: added miiliQ water
July 21stLisza Made glycerol stocks of the 4 pTet-EYFP ligations Kirsten, Alix Mini-prep of pTet-EYFP (3 &4)x2 followed protocol according to Qiagen. Analytic restriction digest of above mini-preps Control used mini prepped pTet from June 24. Added (in order) to 1.5mL micro centrifuge tubes:
Megan, Kirsten, Alix, Lisza Made an 80mL agarose gel for large rig 0.8g agarose 80mL 1x TAE Lanes
Ran at 120V for 40 min( in @ 5:45) stained in ethidium bromide for 10 20 min. Picture: Lisza Made glycerol stocks of maxiprep cultures: 3 lumazine 2 Arg N-term 1 Arg c-term Spun down cultures Lumazine-1=1.35g Lumzine-2= 1.31g Lumazine-3=1.29g Arg c-term=1.26g Arg n-term-1=1.43g Arg n-term-2=1.12g
July 23Did a colony screen PCR: 24 colonies from July 14th DH5α Amp AB+MC+MT plates labeled 10-32 With picked colonies transferred bacteria into a 1.5mL tube with 300µL milliQ water. With same toothpick inoculated 5mL (Amp 5mL) grown overnight at 37°C. Put 1.5L tubes onto heat block at 95°C for 7 min. Centrifuged at full speed for 1 min. Used 5µL supernatant for PCR survey.
Cycle: Colony58 Note: tube 11 contained both samples 11 and 12 July 24th1% agarose gel at 100V of colony PCR Lanes:
PCR was successful, but had negative colonies. No pTet EYFP biobrick, only pTet was in the plasmid July 27Fan & Lisza Preparative restriction digest of pTet-EYFP:
pTet:
EYFP:
Incubated at 37°C for 2 hours Kirsten, Lisza, Mackenzie Purified double digested pTet using QiaQuick PCR purification kit. First run through eluted DNA with 50 µL water then done again and eluted with 30 µL elution buffer. Into -20°C fridge Gel Extraction of EYFP Weight of tube: 1.0973g Weight of tube+gel: 1.9759g Weight gel: 0.8786 Transferred to falcon tube Preformed gel extraction according to Qiaquick Gel Extraction kit. Eluted with 30 µL of elution buffer.
July 28Kirsten, Alix, Lisza Ligation of pTet and EYFP Reaction 1
Reaction 2 (Control)
Incubate at room temperature for 2 hours then transform Restriction Digest of Lumazine 1&2, C-terminal Arg tag, N-terminal Arg tag 1&2.
Incubate at 37°C for 2 hours in water bath. July 28Fan Did a transformation with ligation reactions plus a water control. July 29Running gel of the PCR for EYFP and CFP with c-terminal fusion tags and the restriction digests of n-terminal, c-terminal ARG tags and the lumazine gene. Loading 6µL ladder, 5µL sample+1µL dye Lane:
Did colony PCR of the 5 colonies from the EYFP-1 plater, 5 of the EYFP-2 plate, 4 of the pTet-1, 4 of the pTet-2, one negative control and one positive (EYFP plasmid). Followed protocol of July 23. Megan, Lisza, McKenzie Purify maxipreps using kit. Finished 60 µL of PCR samples with a 5 buffer to 1 PCR sample ratio. Buffer amount = 300 µL. Followed protocol in kit. Ran a gel with ladder, 3 wells for PCR, 3 double wells for extraction
July 30Lisza PCR for fusion c-terminal suffix Control: no template DNA
One for CFP and one for EYFP:
One for CFP and one for GFP:
Control: no polymerase (water= 29µL) Roxanne 1% agarose gel in TAE of the colony PCR E1= plate 1 of EYFP ligation E2= plate 2 of EYFP ligation (-)= no template (+)= EYFP/no pTet P1=plate 1 of pTet control ligation P2=plate 2 of pTet control ligation Lane
Completed the maxipreps of pBAD, TetR, pLacI, mRBS,sRBS and Lumazine 2. They were left at isopropanol precipitation. Restriction Digest dT-EcoRI & XbaI pTet-SAP pSB1A3-1 -EcoRI, Spe1 n-terminal tag-XbaI, PstI Lumazine- XbaI, Spe1 pSB1A3-2- XbaI, PstI C-terminal tag- EcoRI, Spe1
Prefix-N-terminal tag//gene Gene//c-terminal-suffix
Lane:
The PCR worked July 31Mackenzie + Lisza Gel extraction of c-term tag, n-term tag and lumazine.
AugustAug 4Roxanne and Kirsten DNA purification of the 2 pSB1A3 plasmids, dT and pTet using the gel extraction kit Ran gel to check concentrations
Ligations of: pTet-EYFP Lumazine-dT c-term-dT Calculations: EYFP: 3x(878bp [EYFP]/2211bp [pTet])x25ng =29.78ng =4µL EYFP:1 µL pTet 1 µL Lumazine: 1 µL dT 1 µL c-term:1 µL dt
Gel extraction pSBIA3-1 and 2 (plasmid for biobrick parts) Transformation: Tranformed to DH5alpha pBAD = promoter TetR= inverter. Added 2 micro liters of each ligation to DH5alpha. See July 14th Protocol Restrictions of pBAD (SpeI/PstI), TetR (XbaI/PstI), PLacI (SpeI, PstI), sRBS (SpeI/ PstI), mRBS (SpeI/PstI). Used PCR tubes. Each tube contains: 5 micro liters MillQ water, 3 micro liters 10x Tango Buffer, 1 micro liter enzyme 1, 1 micro liter enzyme 2, 20 micro liters of DNA. Put all tubes in thermal controller at 37 degrees. Roxanne, Gel Extraction of pBAD, TetR, pLacI, sRBS, mRBS Lengths: pBAD:2287 bp TetR: 902 bp pLacI: 2279 sRBS: 2092 bp mRBS: 2092 bp Lane:
Aug 5thKirsten and Fan Gel extraction of TetR, pLacI, sRBS, mRBS, pBAD according to Qiagen protocol. Ran analytic gel 1µL Dye + 5µL DNA 6µL Ladder Lane:
tetR concentration: 25ng/µL everything else: 100ngµL
Aug 6Roxanne Set up the following ligations pSB1A3-2+n-term tag and pSB1A3-1+c-term tag
pSB1A3+lumazine
pBAD+tetR inverter
mRBS+n-term tag
Colony PCR of pTet-EYFP, lumazine-dT, c-term-dT Picked 5 colonies of each construct, followed protocol of July 23. E1-E5=EYFP colonies 1-5 E+=EYFP (no pTet) L1-L5=lumazine-dT colonies 1-5 L+= lumazine (no dT) C1-C5= c-term-dT colonies 1-5 C+= c-terminal (no dT) -=no plasmid Lisza Mixed 10mM primers for: antisense suffix primer and FP N-term fusion primer Ashley, Mackenzie, Lisza Analytical gel of colony PCR’s (from earlier) Volumes:
Lane
Transformation of mRBS+n-term, pBAD+tetR, pSB1A3+lumazine, pSB1A3+c-term, pSB1A3+n-term Negative control: water+25 µL DNA Transformed according to iGEM protocol. Plated on ampicillin plates at 100µL and 400 µL volumes and incubated overnight. PCR of EYFP and CFP according to protocol of July 30th. Aug 7The transformations seemed to have all worked, although there are no colonies on plates marked pSBB1A3+N-term and several on the negative control plate. Speculation: tubes were switched or improper labeling. Solution: Colony PCR Lane:
Set up restriction digest of the preperative PCR of EYFP (c-term) and CFP(c-term) fusion proteins DPNI, XbaI,PstI DPNI, EcoRI, speI for the n-term proteins Picked colonies of the transformed cells for pBAD-TetR, pSB1A3 (n-term), pSB1A3 (c-term), pSB1A3 (lumazine) and mRBS-Nterm 3x in ampicillin 5mL tubes.
Aug 11Roxanne Restricted all the minipreps with EcoRI to check the size of the plasmids prior to sending away for sequencing (10µL rxns) Restricted:
in 30 µL rxns to prepare for fusion to CFP of EYFP Setting up ligations of:
mmS6 gene was transformed into DH5α by Megan and Fan, set up a few cultures for minipreps. Aug 13Set up restrictions of :
Gel Extractions of:
Using Qiagen bench top protocol Set up ligations of:
Ran a concentration gel of:
Concentrations:
Ran 1% agarose gel (with 2 wells taped together) Lane:
Ran at 110V for 30min Gel extraction and purification of CheZ and mms6, mmS6 split into two tubes. Following Qiagen kit purification benchtop protocol. Concentration Gel (1% agarose) Lane:
Only 9/18 transformations worked Success:
Added more ligase and will re-transform the following:
Transformed all of the above. Picked colonies of successes. Aug 15thThe transformations didn’t work! Restart Picked 3 colonies of cheZ-dT 1 of mmS6+dT (for mini and maxiprep) 3 of fused C-EYFP 3 of pSB1A3-mmS6 3 of riboswitch in pSB1A3 3 of GFP gene 3 of CFP (no promoter) 2 of the riboswitch and all of the N-EYFP in pSB1A3 turned red suggesting that the RFP reporter was religated in. Will miniprep the “good” cells and restrict with EcoRI to check for size Glycerol stock.
Aug 18Megan and Ashley Pelleted down cells (small samples and large samples) picked from transformations to prep for mini-preps. Add 750 x2 micro liters of your small sample solution to centrifuge tube, Add 50 mL of large sample to large centrifuge tubes. Balanced the Centrifuge and ran for 2 minutes at 13000 rpm. Dump out Supernatant/ leave pellet. Add more sample, centrifuge, and remove more supernatant. Add remainder of sample, centrifuge, remove supernatant. Left samples in -20 degree freezer. Aug 19Kirsten and Mackenzie Maxiprep of fused C-EYFP and mmS6-dT See protocol for July 13th. Aug 20Fan and Mackenzie Miniprep for:
Restriction digest with EcoRI
37°C for 1 hour. Need to run agarose for all restriction digested samples and DNA samples Roxanne, Mackenzie Running a gel of restricted minipreps Checking mini prep concentration with UV spec Lane
Lane
Aug 25Roxanne Set up restrictions 1,4,7 are c-term 2,5,8 are n-term 1,2=EYFP 4,5=CFP 7,8=ECFP
Set up maxipreps for:
Ligations:
Aug 26Lisza Deactivated to reactions @ 65° for 15 min Roxanne Set up liquid cultures of:
Aug 27Fan Miniprep of the O/N culture [DNA] unknown DNA was eluted from the spin column in 50mL milliQ water
Kirsten Gel extraction of GFP, mmS6-dT,CheZ-dT Purified riboswitch, sRBS, c-term-dT, N-term-mRBS. Eluted with 50µL of EB Into -20°C freezer. 10µL ligations of GFP+dT, sRBS+mmS6-dT and riboswitch+cheZ. Left at room temp for 2 hours until transformation. Alix Transformations of ligations following iGEM protocol Aug 31Roxanne PCR of CFP, ECFP, EYFP w/ n-term prefix or c-term suffix 20µL Restrictions of:
SeptemberSeptember 3Roxanne, Ashley, Kirsten Made 1.5L agar LB media according to lab protocol. Roxanne is autoclaving tomorrow. Pick 3 lumazine-dT colonies from glycerol stocks to grow overnight Restriction Digest Reaction 1
Reaction 2
Into HWB at 37°C for 2 hours Gel Electrophoresis (concentration): Lane
5µL sample, 1 µL loading dye (6x) 6 µL ladder Ran at 100V for 1 hour Concentrations good! Alix Gel extraction of: sRBS-mmS6-dT riboswitch-cheZ-dT GFP-dT mmS6 Following Qiagen benchtop protocol September 4Ashley Ligation of:
Reaction
Restriction Digest
September 8Ashley Made 1.5L LB agar according to protocol Made 300mL LB broth according to protocol Gel Electrophoresis (for extraction) 5 µL ladder 5 µL sample, 1 µL dye Lane
^there was a control (no enzyme) but not enough DNA to load Ran at 1000V for one hour Gel extraction according to Qiagen benchtop protocol Sept 10Ashley, Kirsten Ran 1% agarose Lane
3 µL sample + 0.5 µL dye 6 µL 1 kb ladder Mackenzie Transformation into DH5α
The following were not transformed due to lack of competent cells:
September 14Ashley PCR of EYFP, CFP and ECFP See Aug 6 for volumes CFP(1)=prefix primer and antisense C-term suffix CFP(2)=N-term prefix and antisense suffix EYFP(1)=prefix primer and antisense C-term suffix EYFP(2)=N-term prefix and antisense suffix ECFP(1)=prefix primer and antisense C-term suffix ECFP(2)=N-term prefix and antisense suffix Cycle: tail67 Sept 16Ashley Miniprep of mmS6-dT 1,2 and 3. Used Qiagen benchtop protocol and kit. Eluted with 50 µL buffer EB. Put into -20°C freezer orange tray. PCR Purification of restriction digest of PCR products. October |