cosmid purification
QIAGEN
Mari.L.S...@dartmouth.edu (Mari L. Shinohara) wrote:
>I'm having a hard time purifying a cosmid clone.
>I understand that handling cosmids could be sometimes tough. I always
>streak clonies one day before purification, then Terrific Broth was
>used for bacterial propagation. I've got a small amount of cosmid with
>Qiagen only once out of six trials: Usually, I didn't get any.
>I would greatly appreciate if anyone can give suggestions.
Dear Mari,
I have a few helpful suggestions that you may or may not have already
tried. I hope they help.
1. Estimate the culture volume required based on a few pilot experiments
(i.e. preparation by crude methods or small scale preps if you are going to
purify large quantities). The QIAGEN-tip 20 binds ~12-14 ug of cosmid DNA -
the binding capacity is 30-40% less than plasmid because of the larger
size.
2. Use 5 ml of P1, P2 and P3 buffers for every 100 ml of LB culture (do
not use rich broth (TB) or the cell density will be too high for efficient
alkaline lysis Ñ this could lead to lower than expected yields or
inconsistent yields).
3. Follow the recommended protocol for the QIAGEN plasmid kit and also
take note of special instructions of page 24 of the QIAGEN Plasmid Handbook
(Feb 1995).
4. Pre-heat the elution buffer to 50¡C. This helps for larger plasmids >10
kb. Also, after you elute as recommended, repeat elution with another half
volume of elution buffer to make sure you recovered 100%. Do not combine
with the first eluate. Check it first, to see if it actually contains
cosmid DNA Ñ it may all be in the first eluate.
5. During the precipitation and wash step, the DNA pellet is loose and
care should be taken when you remove the supernatant. Also, do not overdry
the pellet, or the DNA will be hard to re-dissolve.
6. Resuspend the DNA in TE, pH 7. If possible, allow the pellet to
re-dissolve at 4¡C overnight. Cosmid DNA takes longer to go into solution
because of its large size.
If you need any further recommendations, please call me at 800-426-8157 or
Technical Service at 800-362-7737.
Sincerely,
Kim Budelier
--
QIAGEN Inc. 800-362-7737
Temporary Email Address qia...@kaiwan.com