Kinases Expression Oct 2012
Plasmids using Nucleobind columns
need to make 1383 (genomic DNA contamination), 1385 (low yield), 1382 (low yield and genomic DNA contamination)
10/15/2012
Plasmid midiprep using used nucleobound Xtra midi column.
Regenerate column with 0.1 M NaCl, 1.5M NaCl, then water. Equilibrate with EQU-EF.
Spin down cells from about 235 ml culture, OD600=2.3.
Resupsend pellet with 8 ml RES-EF, Lyse with 8 ml LYS-EF, incubate at RT for 5 min. Add 8 ml NEU-EF, incubate on ice for 5 min. Spin at 12000 rpm for 5 min. Filter junk, load filtrate to one midi column. Wash filter with 5 ml Buffer FIL-EF, wash with 35 mL Buffer ENDO-EF, then 15 mL Buffer WASH-EF. Elute DNA using 10 ml 5 mL Buffer ELU-EF. Collect 1 ml fractions.
| Fraction | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD260 | 15.3 | 142.4 | 576.6 | 400.7 | 207 | 175.5 | 87.8 | 9.8 | 9.8 | 3.5 |
| 260/280 ratio | 1.43 | 1.83 | 1.91 | 1.86 | 1.87 | 1.85 | 1.85 | 1.7 | - | - |
Combine fractions 2 to 7. Add 4.5 ml isopropanol. Store at 4 oC O/N.
10/16/2012
Midiprep of 1382 using 4 midi prep columns
column1
| Fraction | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD260 | 7.6 | 118.2 | 494.1 | 366.7 | 189.3 | 92.5 | 42.4 | |||
| 260/280 ratio |
Column 2
| Fraction | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD260 | 14.5 | 502.8 | 496.9 | 268.1 | 164.4 | 83.1 | 45.9 | |||
| 260/280 ratio |
Column 3
| Fraction | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD260 | 6.3 | 335.8 | 472.4 | 217.2 | 127.2 | 78.7 | 49.9 | |||
| 260/280 ratio |
Column 4
| Fraction | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD260 | 8.9 | 543 | 554.6 | 427 | 161.3 | 53.9 | 24.7 | |||
| 260/280 ratio |
Spin down plasmid on 10/16/2012
2012-10-31
exprimenter: yingying lu
kinase plasmid QC
| lane | 1 | 2 | 3 | 4 | 5 | 6 |
| Nhe1+Not1 | 1382 | 1383 | 1383 | 1385 | 1386 | 1406 |