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;

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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)

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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