Online life science pharma marketplace and platform for products and services.


Current location: Home » Ask » Molecular biology » Content

no dna in visible colonies

Pending answer Credit reward to answer the question: 0 -
 Hello? I need help for dna purification. I have been doing dna cloning. My vectors are 4.9kb, 5.
5kb, 5.9kb and inserts are 186bp, 184bp, 675bp. I ligated my insert and vector with 3:1 ratio and incubated at 17C for overnight. Next day, half of the ligation mixture transformed to DH5a competent cells. Transformed colonies were visible and I did dna purification using Alkaline solution I, II, III. But there were no dna. i have been suffering this problem with for last 3 weeks. I did this several times by changing LB plate, ampicillin, DH5a cells  and fresh Alkaline solutions. But still there is no dna. I always found genomic DNA and RNA. I also did colony PCR to check that i have the insert but didn't get any bands. i transformed also vector itself to DH5a as a negative control and i couldn't  even get any dna  from vector alone. I am really frustrating with this and please give me some advise?? I really need help :(
total 276 hits     Asked by: Anonymouse I want to answer  

[ AskSearch ]  [ ]  [ Tell a friend ]  [ Print ]  [ Close window ]  [ Return to top ]