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Center for Protein and Nucleic Acid Research

Recommended DNA Purification Protocols

Plasmid Purification: QIAprep Spin Miniprep Kit
http://www1.qiagen.com/Products/Plasmid/SmallScaleKits.aspx

PCR Product Purification: QIAquick PCR purification
http://www1.qiagen.com/Products/DnaCleanup/PcrCleanup.aspx

DNA submission for sequencing

Two modifications are recommended to the above purification protocols.

1. After the PE buffer is added to the column during the wash step, wait 5 minutes before centrifuging. This gives the salts a chance to dissolve in the buffer.

2. Elute and submit your sample in purified H2O, not EB buffer.

Note: Do not elute or quantitate your DNA template in "TE" buffer. The EDTA in TE buffer will cause the cycle sequencing reaction to fail and/or the quantitation to be incorrect.

Desalting samples

If your samples are coming up "blank", a typical problem is too much salt. To desalt your samples we recommend using Amicon-Microcon Spin Columns from Millipore (to order: 1-800-645-5439). Ethanol precipitation can introduce too much salt into the sample, which will cause the cycle sequencing reaction to fail. For Plasmids use the Microcon YM-100 for PCR product you can select from the tables below based on the size of your PCR fragment.

Microconcentrator and Cut-off Qty./Pk Catalogue No.
Microcon YM-3-3,000MW(Yellow )
8/pk
24/pk
100/pk
42420
42403
42404
Microcon YM-10-10,000MW(Green )
8/pk
24/pk
100/pk
42421
42406
42407
Microcon YM-30-30,000MW (Clear)
8/pk
24/pk
100/pk
42422
42409
41410
Microcon YM-50-50,000MW(Rose)
8/pk
24/pk
100/pk
42423
42415
42416
Microcon YM-100-100,000MW(Blue)
8/pk
24/pk
100/pk
42424
42412
42413
Microcon Model Color Code Cut-off MW Nucleotides -- Max g-Force Spin Times -- --
-- -- -- SS1 DS1 -- 4 °C 25°C N2
3 Yellow 3,000 10 10 14,000 185 95 70
10 Green 10,000 30 20 14,000 50 35 20
30 Clear 30,000 60 50 14,000 20 12 10
50 Rose 50,000 125 100 14,000 10 6 6
100 Blue 100,000 300 125 500 24 15 --

1. SS = single-stranded, DS = double-stranded.

2. N = No Control.