PCR-Check, Screen-Check, MiniPrep - HUH?? - PCR product transformed, grown on AMP, no mini (Aug/25/2004 )

Hi - I need some help with getting DNA from my colonies.

I have a PCR product of a vector + insert.
1. Ran PCR product on gel and verified
2. Transform into One Shot Competent cells
3. Grown on LB+AMP plates and LB+Kana(as negative control)
- Grows on AMP doesn't grow on Kana
4. Picked colonies from LB+AMP, grow in liquid LB+AMP
5. Take product and perform mini-prep.
6. Miniprep product shows no DNA!!!????

I've rerun steps 2-6 and still nothing. I took mini-prep product and ran DNA concentration and it shows about 50 ug/ml concentration. But running on the gel shows no product.

I ran again with more product at 65V and only 30 minutes to make sure nothing is running off, still no product.

Any ideas? Thanks!

Hi,

first of all, did u digest the vector and insert accordingly with the RE that you inserted into your primer for the insert during design which is in accordance to the vector sequence. It seems like u did not mention it in ur so called process of performing a ligation. Are u doing a blunt end ligation?

you mention u pcr the vector. how do u do that?

Anyway, after screening and miniprep, usually, u get either a bigger band (containing inserts) or the original plasmid linearized. Getting nothing could mean that the miniprep failed.

Perhaps u want to give more details so that some of us here can help

cheers,
tltan

To get more DNA extraction, maybe u should try spinning down more cultures from the broth. U may try using NB (Nutrient Broth) instead of LB. How much volume did u spin when centrifuging? Make sure there is no contamination.


By the way, what about ur master mix? Are ur calculations right?