Editing Kinases Expression Oct 2012

Plasmids using Nucleobind columns 

[[Image:Plasmid maxi 10 14 2012.jpg]] 

[[Image:Plasmid maxi 2 10 14 2012.jpg]] 

need to make 1383 (genomic DNA contamination), 1385 (low yield), 1382 (low yield and genomic DNA contamination) 

10/15/2012<br> 

Plasmid midiprep using used nucleobound Xtra midi column.<br> 

Regenerate column with 0.1 M NaCl, 1.5M NaCl, then water. Equilibrate with EQU-EF.<br> 

Spin down cells from about 235 ml culture, OD600=2.3.<br> 

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&nbsp; 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.<br> 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 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.<br> 

10/16/2012<br> 

Midiprep of 1382 using 4 midi prep columns 

column1 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 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 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 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 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 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 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 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 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

<br> 

<br> Spin down plasmid on 10/16/2012 

[[Image:1383-1406 maxiprep digestion.JPG]]<br> 





2012-10-31

exprimenter: yingying lu

kinase plasmid QC

[[Image:Kinase_plasmid_QC_11-1-2012.JPG]][[Image:Kinase_plasmid_QC2_11-1-2012.JPG]]

{| width="232" height="45" cellspacing="1" cellpadding="1" border="1"
|-
| lane
| 1
| 2
| 3
| 4
| 5
| 6
|-
| Nhe1+Not1
| 1382
| 1383
| 1383
| 1385
| 1386
| 1406
|}



[[Category:Protein_Expression]]