Screening Tranformation Colonies: Funny Gel Pic and no plasmid?!?! - (May/19/2008 )
QUOTE (tfcheng @ Oct 9 2008, 12:50 PM)
QUOTE (Hanming86 @ Oct 8 2008, 05:11 AM)
QUOTE (tfcheng @ Oct 7 2008, 10:22 AM)
Hi jeff and everyone,
I wonder if you have solved your mystery? especially the part that culture grow slow after O/N incubation, because I am facing the same problem here. I am doing a site-directed mutagenesis by following quick-change protocol from Stratagene. After DpnI treatment, I transformed reaction into GC10 competent cells, grow O/N on carb/agar plate. Examine the plate next day and the colonies look fine. Pick them out and grow in LB with selection at 37C, O/N, the culture look very dilute, and I only got a small bacterial pellet. Use Qiagen miniprep kit and got no plasmid at all.
Hope some help!!
I wonder if you have solved your mystery? especially the part that culture grow slow after O/N incubation, because I am facing the same problem here. I am doing a site-directed mutagenesis by following quick-change protocol from Stratagene. After DpnI treatment, I transformed reaction into GC10 competent cells, grow O/N on carb/agar plate. Examine the plate next day and the colonies look fine. Pick them out and grow in LB with selection at 37C, O/N, the culture look very dilute, and I only got a small bacterial pellet. Use Qiagen miniprep kit and got no plasmid at all.
Hope some help!!
Slow growth normally imply 1 . ur antibiotic conc is too high or 2. ur culture got no plasmid ( some residual growth are sometimes expected especially when u use carbenicillin as ur antibiotic or any bacteriostatic type antibiotic). DID you do a control of " no plasmid" to check for the strength of your antibiotic. u DIDNOT mention anythiong about control. Experiment w/o control = experiment w/o direction
Good idea!! But I think if I need a control to know if the [carb] is too high, I should transform bugs with the same vactor that I used to do the cloning. Well, I guess Experiment w/o logic = Experiment w/o brain.
I'm just suggesting possible causes in general and not really in your particular case. Besides, the assumption is made NOT based on ur transformation result but the next " SUBCLONING" step... in your case you should know fully well that it's a " no plasmid" scenario right away because you have the obvious proof already
I thought u knew but oh well =.="experiment w/o logic = experiment w/o brain" nice 1 but quite harsh , like just saying if you don't do ur experiment right, you're stupid problem is we do mistake all the time .haha. trying to mock everyone in the scientific community?
And just so u know ( i guess i need to underscore "IN YOUR TRANSFORMATION STEP"), having growth on the plate and u NOT doing a control of "NO plasmid" shows no proof that your transformation is 100% working till u proceed to plasmid extraction step or colony lysis PCR which u did the former and found out to disappoint u.
Think , what if u have the same growth phenotype on the plate as well when u did " NO plasmid" control. would u still want to proceed?!
that's the logic...
i just want to emphasize the importance of a control. it makes ur confidence rise 100% when u see ur result.
but if u're the type who don't like to care about ur result ,,, let it be let it be ... what can i do ..
-Hanming86-