Hi,

How much ug of DNA do you use to transform. Have you tried with any positive control? Also, when you plate down, how much ul do you use from the total amount?

You should be careful if there is any contamination in your competent cells. Please illustrate your result. Thanks.

I used 3 ul of ligation mixture of 1:3 molar ratio containing 100 ng of pET15b. I plated 200 ul of outgrowth culture after 1 hour incubation at 37 C. All controls are good there is no growth in transformed cut pET15b while pET15 intact is showing good colonies only two plates containing inserts are showing comet like colonies.

I think maybe the DNA is contaminated with other colony. You can try streaking your positive construct and select from it. Then proceed one more time for transformation.

Hi,

I agree with Pratibha Srivastava and suggest that you repeat the experiment of PCR amplification and DNA fragments extraction for subsequent ligation and transformation experiments.

Good luck!

Dear Sandeep.

Did you use one or more restriction enzymes? What is the fragment size? What sort of transforming DH5alpha are you using (chemo-competent or electro ones? What exactly are you calling streaky colonies? Could you send a pic? Have you already solved it? If not and you feel ok to share a few more info, I would like to try to help you.

All the best,

Adriano Alcântara

Hello Adriano Costa de Alcantara I used Ndei and XhoI and my insert size is ~3.7 kb. I,m using CaCl2 treated competent cells and using heat shock method, 42 oC for 45 sec. Streaky colonies means a few colonies are sticking togethers and appearing like a comet shape.

Hi, Sandeep.

Sorry for the delay.

How long did you let your colonies grow to collect them? Are they comet shaped the day after streaking it?

If you're leaving it for a few days, even in the refrigerator, they may start to grow as satellite colonies. (Check google images using the following terms: bacterial satellite colonies). Seems like your images???