The copy number of pPICZ alpha A - (Dec/26/2011 )

Hi everyone!
I,m currently working with pPICZalphaA vector. After the transformation, I did plasmid isolation using phenol/chloroform method. when run a gel I couldnt find a expected size of band.. If someone work with the same plasmid please help me...
Thanks

Did you measured DNA before loading. And did you got any band on the gel.

What host strain did you use? ...as far as i remember pPICZ has a Zeocin resistance!

Any E. coli strain that contains the complete Tn5 transposable element (i.e. DH5αF´IQ, SURE, SURE2) encodes the Zeocin resistance gene (bleo). These strains will confer resistance to Zeocin. For the most efficient selection, use an E. coli strain that does not contain the Tn5 gene (i.e. TOP10, DH5, DH10, etc.).

...so it could be that you got false positive clones?

Regards,
p

thank u to replay me...
@ Biouday:- yep my concentration of DNA is 50ng/microlitr. and also i got 2 bands regarding to plamid. Yet the both bands are very faint. Is thislow copy no. vector?

@ pDNA: Ya it is a zeocin resistant vector, and i used E.coli DH alpha to transformation.

no, it has a pUC origin and therefore should be high copy!

what size of bands do you get? ...did you consider that plasmid DNA can run in different confomations (supercoiled, linear, open circle)?

can you shortly comment on your protocol for plasmid DNA preperation?

Regards,
p

@pDNA: ya, I didn't run with marker to check the size, I just ran to make sure there is plasmid..

Ya there were 2 bands, it was due to supercoiled and open super coiled structure nah?
this is i follow..

1.5ml of culture was centrifuged, 150miclitr of ET buffer(Tris HCL ph 8.0, EDTA, RNAse) added to pellet, resuspended and allowed 3-4 mins in RT.
150 miclitr of lysis buffer ( NaOH, SDS) was added and mixed by inverting and allowed 5 min at RT.
150miclitr of KAc solution (KAc, gl HAc & Water) was added, inveted and allowed in ice for 5 min.
Spun it at 12000/ 5 min, removed the supernatant to new tube and equal volume of Phenol/chloroform was added and vortex for 10 sec.
Spun it at 12000/ 5 min, removed the supernatant to new tube and equal volume of chloroform was added.
Spun it at 12000/ 5 min, removed the supernatant to new tube, 2 volume of absolute ethanol was added and kept on Ice for 30 min.
Spun it at 12000/ 10 min, removed the ethanol and washed it using 70% ethanol and allow to air dry.
then 50 micrlitr of TE buffer was used to dissolve the pellet and run on a gel.
.............

your protocol seems reasonable!

i'm a bit confused ...you said that:

afRNA on Tue Dec 27 16:20:00 2011 said:

in your first posting you said:

afRNA on Mon Dec 26 17:15:53 2011 said:

how could you say that the bands did not had the expected size if you didn't run a marker?

I'm not sure what your real problem is? ...you have the wrong plasmid/no plasmid/to low plasmid conc.?

Regards,
p