Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biochemistry Forum > DNA Forum
Register Search Today's Posts Mark Forums Read

DNA Forum Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.

pCDFduet-1 plasmid dialemma

pCDFduet-1 plasmid dialemma - DNA Forum

pCDFduet-1 plasmid dialemma - Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.

LinkBack Thread Tools Display Modes
Old 07-04-2010, 10:04 AM
Pipette Filler
Points: 59, Level: 1 Points: 59, Level: 1 Points: 59, Level: 1
Activity: 0% Activity: 0% Activity: 0%
Join Date: Jul 2010
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default pCDFduet-1 plasmid dialemma

Has anyone had problems with pCDFduet-1 uptake by E. coli cells?

I am currently trying in vain to do coecpression of two proteins using the pCDFduet-1plasmid (to use one protein to improve the solubility of another). This plasmid contains spectinomycin antibiotic resistance. I have grown E. coli transformed with the pCDFduet-1 plasmid on agar containing freshly prepared spectinomycin solution (either 50mg/ml or 100mg/ml) [1:1000; antibiotic solution:agar]. The cells grow fine, and due to the antibiotic resistance, theoretically, only cells which have taken up the plasmid should grow. However, when I carry out a digest or PCR, no evidence of the plasmid is seen on the 1% agarose gel I run afterwards. I always run a positive and a negative control on the gel. The positive control always displays evidence of a plasmid, yet the samples mimic the negative control.

If anyone has any suggestions to explain this riddle (i.e. cell growth despite no plasmid with antibiotic resistance / causes of either no plasmid uptake or plasmid uptake but no evidence on the gel). Or any other bright ideas for coexpression of two proteins that is likely to work, Id be very grateful.

Many thanks

Reply With Quote
Old 08-11-2010, 07:21 AM
Pipette Filler
Points: 563, Level: 11 Points: 563, Level: 11 Points: 563, Level: 11
Activity: 0% Activity: 0% Activity: 0%
Join Date: Apr 2010
Posts: 18
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: pCDFduet-1 plasmid dialemma

I'm not so familiar with the pCDFduet-1 plasmid or spectinomycin, but if I'm not mistaken, spectinomycin is similar to streptomycin? Certain point mutations in the rpsL gene can confer resistance to streptomycin, and perhaps this is the case with spectinomycin as well.

Which strain are you using? Have you checked that is is sensitive to streptomycin? How many transformant colonies do you get? If you only get a few (<10), these could simply be spontaneous mutants of rpsL and your transformation failed for some reason. But you imply that you are getting lots of transformants, so maybe the strain is already resistant? You could also switch plasmids and use one of the other pETduet vectors. I've had good success with pETDuet-1 (ampicillin resistance).
Reply With Quote

dialemma , pcdfduet1 , plasmid

Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
plasmid re-ligation problem Vagelis Protocols and Methods Forum 4 06-20-2012 08:29 PM
problem with pWATERGATE plasmid ismawanto Molecular Cloning Forum 5 05-31-2010 10:30 AM
Plasmid Preps from MRSA Anthony C. Hilton Protocols and Methods Forum 1 07-10-2004 12:04 AM
yeast plasmid preps johnspar@earthlink.net Yeast Forum 0 02-20-2004 02:13 AM

All times are GMT. The time now is 01:28 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12760 seconds with 16 queries