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| | == Notebook: Week 10 == | | == Notebook: Week 10 == |
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| - | ''Friday''
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| - | - Planned/mapped out next 4 weeks
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| - | - Took dialyzed bag with purified eGFP-RiAFP and divided it into 12 eppendorfs, containing 1 mL each. Flash-froze tubes in liquid nitrogen, then quickly used a hot edge to poke holes into top of eppendorfs to allow sublimation. Tubes then placed in lyophilizer for 3 nights.
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| | | | |
| - | ''Saturday''
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| - | - Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?):
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| - | - Digested 6 constructs and linearized pSB1C3 with EcoRI, PstI
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| - | - Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays
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| - | - Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI
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| - | - Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)
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| - |
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| - | ''Sunday''
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| - | - Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074
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| - | - Transformed ligations into DH5alpha cells, plated them
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| - |
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| - | ''Monday''
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| - | - Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below):
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| - | - Picked up lyophilized cells; resuspended first in 50 mL of 50 mM Tris-HCl (pH 7.5); A280 ~ .72, A488 ~ .13; however, looked cloudy (possible aggregation?), so centrifuged at 13K rpm for 10 minutes, and UV-vised the supernatant, A280 ~ .23, A488 ~ .04, suggesting around 75% was aggregated. Tried again to resuspend in 100 mL Tris-HCl and adding 50 mM NaCl; still no good; tried adding acid and killed protein activity
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| - |
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| - | ''Tuesday''
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| - | - Miniprepped successful colony PCRs and submitted samples for sequencing
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| - | - Prepared more buffers for protein purification
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| - | - Transformed eGFP and eGFP-RiAFP and RiAFP cells
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| - | ''Wednesday''
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| - | - Protein purification round 2!
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| - | - Prepared a lysis buffer of 50 mM NaH2PO4, 300 mM NaCl (with 1 mM PMSF added before lysis), and 500 mM imidazole elution buffer (with same salts as lysis buffer)
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| - | - Innoculated eGFP, eGFP-RiAFP, and RiAFP cells
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| - | - Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer
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| - | - Sonicated (using Spiegel lab sonicator) with 1 s on/4 s off for 25 total minutes of sonication for each resuspended pellet (unforunately, RiAFP sample by itself was foaming when we returned...)
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| - | - Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump
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| - | - Spin-concentrated samples using
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| - | - Ran w FPLC/Fraction Collector Ni-NTA
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| - |
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| - | ''Thursday''
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| - | - Ran/checked gels with lots of protein
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| - | - Grew up more cells
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| - | - Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration)
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| - | - Incubated with TEV
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| - |
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| - | ''Friday''
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| - | - Size exclusion of TEV-ed protein
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| - | -
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| | </div> | | </div> |
| | <div style="clear:both"></div> | | <div style="clear:both"></div> |
| | </div> | | </div> |
"
