Plasmid Kit comparison
miniprep kit comparison
by Haiyan Lei
Make miniprep from bacteria grown in individual culture tube (4 ml)
| 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;
==============
7/28/2011
by Haiyan Lei
Make miniprep from bacteria grown in individual culture tube (4 ml)
| 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).
--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
| 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
elute DNA with 50 µl water, load 5ul per well into gel
| 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
5 µl run in 1% gel
1kb ladder used
| 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
| 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
5 µl run in 1% gel
1kb ladder used
Eluted DNA with 50 µL water (lanes 7-10)
| 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 |