Team:Lethbridge/Notebook
From 2009.igem.org
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.
September 24Ashley, Kirsten 1% gel electrophoresis 6uL ladder (1kb) 5uL sample +1 uL 6x loading dye 100V, 40min Lane
Gel was run to check for DNAses, found that DPN-1 had DNAses. Sept 251% agarose gel 100V, 40min Lane:
Only pLacI success, gell extracted and eluted with 50uL buffer EB. Miniprep of:
Restricted all of the above with EcoRI
for 1 hour. Restriction of fusion proteins with either EcoRI,SpeI or XbaI,PstI
Ran on a gel (1% agarose)100V,40min Lane:
Lane:
Setup Liquid culture of the Mr. Gene mmS6 x3
September 28Ashley 1% agarose gel
Lane:
Ran at 100V for 40 min OctoberOctober 1Roxanne Gel for gel extraction Restriction Ribo-Chez-dT
Ligation
Aliquoted DH5α cells Ashley Gel Extraction of lumazine-dT
Eluted with 50uL buffer EB Ligation
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