Editing Plasmid Kit comparison

miniprep kit comparison 

by Haiyan Lei<br> 

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

[[Image:Kit comparison.JPG]]<br> 

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

<br> 

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; <br> 

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

==============<br> 

7/28/2011 

by Haiyan Lei 

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

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

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

<br> load 5 µl to 1% gel. <br> 

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) 

==============<br> 

Test different miniprep kits<br> 

By Yolanda Zhang<br> 

Use 5 ml bacteria from '''the same&nbsp; culture flask '''to make miniprep<br> 

Use fresh P1 buffer 

run 5 ul on 1% gel<br> 

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

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

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

<br> 

Conclusion: Omega kit is the best.<br> 

===============<br> 

8/1/2011 <br> 

Test different column using Omega buffers<br> 

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

use fresh P1 buffer 

This is done by David Hu <br> 

<br> [[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 #<br> 
| OD260 µg/ml<br> 
| Total Volumn µl<br> 
| Total Amount<br> 
| 260/280<br> 
| Column<br> 
| Buffer <br>
|-
| 1<br> 
| 102.2 
| 50<br> 
| 5.11 
| 1.83<br> 
| MN 
| Omega<br>
|-
| 2<br> 
| 89.6<br> 
| 100<br> 
| 8.96 
| 1.79<br> 
| Intron 
| Omega<br>
|-
| 3<br> 
| 131.4<br> 
| 100<br> 
| 13.14<br> 
| 1.62<br> 
| Qiagen 
| Omega<br>
|-
| 4<br> 
| 145.6<br> 
| 50<br> 
| 7.28 
| 1.65 
| Original 
| Omega<br>
|-
| 5<br> 
| 138.9 
| 50<br> 
| 6.945<br> 
| 1.74<br> 
| Omega 
| Omega<br>
|}

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

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

============<br> 

Test different column using Qiagen buffers<br> 

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

use fresh P1 buffer 

by Mindy Chen<br> 

[[Image:Qiagen Mini Prep Gel CORRECT.jpg]]<br> 

5 µl run in 1% gel<br> 

1kb ladder used 

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

<br> 

=============<br> 

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

By Robert Cai.<br> 

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

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

Add 5 µl loading buffer, run 25 µl on gel <br> 

<br> 

[[Image:Mini Prep PCR Purified.jpg]]<br> 

<br> 

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

<br> 

=================<br> 

Used different columns using Qiagen versus Omega buffers.<br> 

By Yolanda Zhang<br> 

Plasmid is plenti-mkate2<br> 

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

<br> 

5 µl run in 1% gel 

1kb ladder used <br> 

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

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

<br> 

[[Category:Protocols]]