|
|
(One intermediate revision not shown) |
Line 24: |
Line 24: |
| <div id="entry"> | | <div id="entry"> |
| | | |
- | == Notebook: Week 10 ==
| + | <h1>Week 11: July 24-31, 2011</h1> |
- | ''Friday''
| + | <ul> |
- | - Planned/mapped out next 4 weeks | + | <li>Monday:<ul> |
- | - 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.
| + | <li>Regenerate Nickel columns</li> |
- | | + | <li>Incubate sample with beads</li> |
- | ''Saturday''
| + | <li>Survival assay</li> |
- | - Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?):
| + | </ul> |
- | - Digested 6 constructs and linearized pSB1C3 with EcoRI, PstI
| + | Tuesday:<ul> |
- | - Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays
| + | <li>Survival Assay again</li> |
- | - Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI
| + | <li>Elution from loose beads + SDS</li> |
- | - Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)
| + | </ul></li><li> |
- | | + | Wednesday:<ul> |
- | ''Sunday''
| + | <li>Run flow-through through beads again</li> |
- | - Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074
| + | </ul></li><li> |
- | - Transformed ligations into DH5alpha cells, plated them
| + | Thursday:<ul> |
- | | + | <li>Researched ice-affinity purification</li> |
- | ''Monday''
| + | <li>Dialyzed sample</li> |
- | - Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below):
| + | </ul></li><li> |
- | - 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
| + | Friday:<ul> |
- | | + | <li>Planned/mapped out next 4 weeks</li> |
- | ''Tuesday''
| + | <li>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.</li> |
- | - Miniprepped successful colony PCRs and submitted samples for sequencing
| + | </ul></li><li> |
- | - Prepared more buffers for protein purification
| + | Saturday:<ul> |
- | - Transformed eGFP and eGFP-RiAFP and RiAFP cells
| + | <li>Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?) by digesting those 6 constructs and linearized pSB1C3 with EcoRI, PstI |
- | | + | </li> |
- | ''Wednesday''
| + | <li>Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays</li> |
- | - Protein purification round 2!
| + | <li>Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI</li> |
- | - 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)
| + | <li>Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)</li> |
- | - Innoculated eGFP, eGFP-RiAFP, and RiAFP cells
| + | </ul></li><li> |
- | - Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer
| + | Sunday:<ul> |
- | - 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...)
| + | <li>Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074</li> |
- | - Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump
| + | <li>Transformed ligations into DH5alpha cells, plated them</li> |
- | - Spin-concentrated samples using
| + | </ul></li> |
- | - Ran w FPLC/Fraction Collector Ni-NTA
| + | </ul> |
- | | + | |
- | ''Thursday''
| + | |
- | - Ran/checked gels with lots of protein
| + | |
- | - Grew up more cells
| + | |
- | - Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration)
| + | |
- | - Incubated with TEV
| + | |
- | | + | |
- | ''Friday''
| + | |
- | - Size exclusion of TEV-ed protein
| + | |
- | -
| + | |
- | | + | |
| | | |
| </div> | | </div> |
| <div style="clear:both"></div> | | <div style="clear:both"></div> |
| </div> | | </div> |