Paul.xu at 14:57, 1 November 2012

2012-11-01T14:57:01Z

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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 

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. 

{| 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. 

10/16/2012 

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
|
|
|
|
|
|
|
|
|
|
|}

 

 Spin down plasmid on 10/16/2012

[[Image:1383-1406 maxiprep digestion.JPG]] 

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]]

Kinases Expression Oct 2012 - Revision history

Paul.xu at 14:57, 1 November 2012