Hylei at 14:29, 4 August 2011

2011-08-04T14:29:55Z

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miniprep kit comparison

by Haiyan Lei 

Make miniprep from bacteria grown in individual culture tube (4 ml) 

[[Image:Kit comparison.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Lane # 
| Plsmid 
| MiniPrep Kit 
| OD260 
| Total Volumn 
| Total Amount 
| OD260/280 
|-
| 1 
| MW 
| 
| 
| 
| 
| 
|-
| 2 
| dsAAV_SMN1 
| Qiagen 
| 30 
| 50 
| 
| 2.02 
|-
| 3 
| dsAAV_SMN1 
| Original 
| 330 
| 50 
| 
| 1.19 
|-
| 4 
| dsAAV_SMN1 
| Omega 
| 10 
| 50 
| 
| 
|-
| 5 
| dsAAV_SMN1 
| Introbio 
| 40 
| 50 
| 
| 1.3 
|-
| 6 
| dsAAV_SMN1 
| Qiagen 
| 
| 
| 
| 
|-
| 7 
| dsAAV_SMN1 
| Original 
| 
| 
| 
| 
|-
| 8 
| dsAAV_SMN1 
| Omega 
| 
| 
| 
| 
|-
| 9 
| dsAAV_SMN1 
| Introbio 
| 
| 
| 
| 
|-
| 10 
| 
| Qiagen 
| 
| 
| 
| 
|-
| 11 
| 
| Original 
| 
| 
| 
| 
|-
| 12 
| 
| Omega 
| 
| 
| 
| 
|-
| 13 
| 
| Introbio 
| 
| 
| 
| 
|}

 

5 µl/50µl run gel,

Kit order: Qiagen, Original, Omega, introbio

Lane 1: Marker

Lane 2-5: dsAAV_SMN1

lane 2, 30 ng/µl, 260/280 is 2.02;

lane 3, 330 ng/µl, 260/280 is 1.19;

lane 4, 10 ng/µl, 260/280 is ---;

lane 5, 40 ng/µl, 260/280 is 1.3;

Lane 2-5: dsAAV_SMN2,

lane 6, 30 ng/µl, 260/280 is 2.02; 

[[Image:Plasmid kit comparison 2 7 27 2011.JPG]]

============== 

7/28/2011

by Haiyan Lei

Make miniprep from bacteria grown in individual culture tube (4 ml) 

[[Image:Plasmid kit comparison 7 28 2011.jpg]]

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Lane # 
| Plasmid DNA 
| Plasmid Kit 
|-
| 1 
| MW 1kb ladder 
| 
|-
| 2 
| pET8XHis-Cre 
| MN
|-
| 3 
| pET8XHis-Cre 
| Original 
|-
| 4 
| pET8XHis-Cre 
| Qiagen
|-
| 5 
| pET8XHis-Cre 
| Omega
|-
| 6 
| pET8XHis-Cre 
| Intron
|-
| 7 
| dsAAV-SMN2 
| MN 
|-
| 8 
| dsAAV-SMN2 
| Original 
|-
| 9 
| dsAAV-SMN2 
| Qiagen 
|-
| 10 
| dsAAV-SMN2 
| Omega 
|-
| 11 
| dsAAV-SMN2 
| Intron 
|-
| 12 
| MW 1kb ladder 
| 
|}

 load 5 µl to 1% gel. 

Mistakes was made because no DNA in lane 5 and 10 (Omega kit )

There are quite a lot of RNA in lan 6, 7, 11, possibly P1 buffers are old (RNase is not working).

--[[User:Hylei|Hylei]] 16:59, 28 Jµly 2011 (EST)

============== 

Test different miniprep kits 

By Yolanda Zhang 

Use 5 ml bacteria from '''the same  culture flask '''to make miniprep 

Use fresh P1 buffer

run 5 ul on 1% gel 

[[Image:Plasmid kits comparison 1 7 30 2011.jpg]] 

[[Image:Plasmid kits comparison 1 7 30 2011.jpg]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 566px; height: 289px;"
|-
| Lane # 
| OD260 µg/ml 
| Total Amount 
| 260/280 
| Plasmid 
| Kit
|-
| 1 
| 11.3, 50 µl 
| 0.565 µg 
| 1.72 
| pET30-8XHis-Cre 
| MM
|-
| 2 
| 13.3, 50 µl 
| 0.665 µg 
| 1.82 
| pET30-8XHis-Cre 
| Home Made
|-
| 3 
| 32.7, 50 µl 
| 1.635 µg 
| 1.73 
| pET30-8XHis-Cre 
| Qiagen
|-
| 4 
| 45.4, 50 µl 
| 2.27 µg 
| 1.80 
| pET30-8XHis-Cre 
| Omega
|-
| 5 
| 30.7, 50 µl 
| 1.535 µg 
| 1.91 
| pET30-8XHis-Cre 
| Intron
|-
| 6 
| 20.6, 50 µl 
| 1.03 µg 
| 1.78 
| pAAV-MCS 
| MM
|-
| 7 
| 18.5, 50 µl 
| 0.925 µg 
| 1.83 
| pAAV-MCS 
| Home Made
|-
| 8 
| 23.3, 50 µl 
| 1.165 µg 
| 1.79 
| pAAV-MCS 
| Qiagen
|-
| 9 
| 125.6, 100 µl 
| 12.56 µg 
| 1.79 
| pAAV-MCS 
| Omega
|-
| 10
| 140.0, 100 µl 
| 14 µg 
| 1.86 
| pAAV-MCS 
| Intron
|}

 

Conclusion: Omega kit is the best. 

=============== 

8/1/2011 

Test different column using Omega buffers 

5 ml bacteria from the same culture, plasmid is pAAV-MCS. 

use fresh P1 buffer

This is done by David Hu 

 [[Image:MiniprepComparison 8 1 2011.JPG]]

elute DNA with 50 µl water, load 5ul per well into gel

{| cellspacing="1" cellpadding="1" border="1" style="width: 566px; height: 289px;"
|-
| Lane # 
| OD260 µg/ml 
| Total Volumn µl 
| Total Amount 
| 260/280 
| Column 
| Buffer 
|-
| 1 
| 102.2
| 50 
| 5.11
| 1.83 
| MN
| Omega 
|-
| 2 
| 89.6 
| 100 
| 8.96
| 1.79 
| Intron
| Omega 
|-
| 3 
| 131.4 
| 100 
| 13.14 
| 1.62 
| Qiagen
| Omega 
|-
| 4 
| 145.6 
| 50 
| 7.28
| 1.65
| Original
| Omega 
|-
| 5 
| 138.9
| 50 
| 6.945 
| 1.74 
| Omega
| Omega 
|}

Conclusion: Intron and Qiagen column have higher binding capacity when using Omega buffers 

Problem: why Omega column has lower DNA amount than Yolanda got? 

============ 

Test different column using Qiagen buffers 

5 ml bacteria from the same culture, plasmid is pAAV-MCS. 

use fresh P1 buffer

by Mindy Chen 

[[Image:Qiagen Mini Prep Gel CORRECT.jpg]] 

5 µl run in 1% gel 

1kb ladder used

{| cellspacing="1" cellpadding="1" border="1" style="width: 612px; height: 158px;"
|-
| Lane# 
| OD260 µg/ml 
| Volumn µl 
| Total Amount 
| 260/280 
| Column 
| Buffer Kit 
|-
| 1 
| 63.7 
| 50 
| 3.185 
| 1.80 
| MN 
| Qiagen 
|-
| 2 
| 44.2 
| 50 
| 2.21 
| 1.78 
| Intron 
| Qiagen 
|-
| 3 
| 109.8 
| 50 
| 5.49 
| 1.77 
| Qiagen 
| Qiagen 
|-
| 4 
| 56.1 
| 50 
| 2.805 
| 1.80 
| Original 
| Qiagen 
|-
| 5 
| 97.4 
| 50 
| 4.87 
| 1.74 
| Omega 
| Qiagen 
|}

 

============= 

Use different column to recover DNA from gels using Qiagen QG buffer. 

By Robert Cai. 

Dissolve DNA fragments in gel with QG buffer, load into spin column 

wash with 2 X 350 µl PE, spin 1 min, elute DNA using 20 µl water. 

Add 5 µl loading buffer, run 25 µl on gel 

 

[[Image:Mini Prep PCR Purified.jpg]] 

 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Lane # 
| Mini Prep Column 
|-
| 1 
| MN 
|-
| 2 
| Intron 
|-
| 3 
| Qiagen 
|-
| 4 
| Original 
|-
| 5 
| Omega 
|}

 

================= 

Used different columns using Qiagen versus Omega buffers. 

By Yolanda Zhang 

Plasmid is plenti-mkate2 

[[Image:Plasmid kit comparison 8 3 2011.JPG]] 

 

5 µl run in 1% gel

1kb ladder used 

Eluted DNA with 50 µL water (lanes 7-10) 

{| cellspacing="1" cellpadding="1" border="1" style="width: 729px; height: 228px;"
|-
| Lane # 
| OD260 (µg/ml) 
| Total Volume (µl) 
| Total Amount 
| 260/280 
| Column 
| Buffer Kit 
|-
| 1 
| 130.7 
| 50 
| 6.535 
| 1.21 
| MN 
| Qiagen 
|-
| 2 
| 48.1 
| 50 
| 2.405 
| 1.75 
| Original 
| Qiagen 
|-
| 3 
| 58.3 
| 50 
| 2.915 
| 1.80 
| Qiagen 
| Qiagen 
|-
| 4 
| 109.0 
| 50 
| 5.45 
| 1.78 
| Omega 
| Qiagen 
|-
| 5 
| 35.4 
| 50 
| 1.77 
| 1.70 
| Intron 
| Qiagen 
|-
| 6 
| 146.1 
| 50 
| 7.305 
| 1.65 
| MN 
| Omega 
|-
| 7 
| 79.1 
| 100 
| 7.91 
| 1.74 
| Original 
| Omega 
|-
| 8 
| 93.4 
| 100 
| 9.34 
| 1.83 
| Qiagen 
| Omega 
|-
| 9 
| 99.6 
| 100 
| 9.96 
| 1.83 
| Omega 
| Omega 
|-
| 10 
| 124.0 
| 100 
| 12.4 
| 1.78 
| Intron 
| Omega 
|}

 

[[Category:Protocols]]

Plasmid Kit comparison - Revision history

Hylei at 14:29, 4 August 2011