i need help : lambda phage excision problem !! - labmbda phage excision problem (Aug/16/2005 )
Do you guys face any problems on phagemid excision? Now i m trying to do phagemid excision (Zap express predigested vector Kit). I went through ligation, packaging, Amplification libraries, But !! the last step.. Excision !! i got nothing. No colony grew on my Kanamycin LB plate.
I checked for all of these:
1. blue-white plaques test show a lot of recombinants within my library.
2. i have no problem with Helper phage, the titer is around 10-9 pfu/ml
3. No problem with Bacterial host (XLOLR for excision), they grow well on LB plate without Kanamycin
4. my lambda phage libraries are very good.. around 10-6 pfu/ml
I tried to solve problems by postpone the incubation time for the Bacterial host to express kanamycin resistant after infected with phage.. anyway i still have the same problem. Not any single colony on my Kana-plate.. Any suggestions? Please Please help me.
I have had problems with this. Make sure you use cells which have been freshly streaked, and maximise everything in the protocol, i.e. longest incubation timt, plate largest amount. The cells are highly important, make sure that they are still in log phase - OD 600 of 0.5-1. An OD of 0.7 I have often found to work best.
Hope this helps a bit.
Thank you !! for your quickly reply !! AXOLOTL !!
I quite sure for the freshly streak Cell. I did it again yesterday
i still haven' t got any colonies.
anyway i ll try your suggestion today again.
I have a few questions:
1. I read the trouble shooting about excision, the manual suggest only "be strick about the ratio between 'Lambda Phage : Host Cell (for amplification prior excision with XLOLR strain) : Helper Phage' . It should be 1:10:100
Why? if Lambda Phage and Helper phage is efficient, they should co-infect the Host in a good rate. No matter how many cells are in the reaction. Did i think in the right way? What do you think? Did you strick with it?
2. Last night, i postponed the incubation time after i added XLOLR strain (for the last step, excision) from 45 min to 60 min. and still get nothing.
So.. today, i think i m gonna postpone to 90 min. What do you think? THe longer time may cause any undesirable result?
Thank you very much
Make sure you are using the ExAssist helper phage and not R408.
Help maguro!! i'm facing the exact problem that u've faced 3 years ago!! I've tried every possible option to troubleshoot it. Prolonged incubation, adding rich medium before plating on antibiotic plate..i've reach to the point of all hope lost already! and i'm extremly down about it!
May i know how do you overcome all these?