Problems with Transformation - (Jul/24/2006 )
Freshman:I'm doing Transformation procedure...
So,What is blue & white screening?Is it white colonies surrounding blue colonies and vice versa?
What is the significance of the blue & white colonies?
If i use kanamycin instead of ampicilin,will i get satellite colonies?
Why i will get satellite colonies instead of main colonies?
What is the significance of the blue & white colonies?
If i use kanamycin instead of ampicilin,will i get satellite colonies?
Why i will get satellite colonies instead of main colonies?
I copied them from promegas site:
What is blue/white screening and how is it performed?
The pGEM®-Z Vectors possess a multiple cloning region within the alpha-peptide coding region for the enzyme beta-galactosidase (LacZ gene). Insertional inactivation of the alpha-peptide allows recombinant clones to be identified by color screening on indicator plates. Vectors without an insert express a functional beta-galactosidase enzyme when expressed in bacteria which contain an episomal or chromosomal deletion in the lacZM15 gene, which results in inactivation of the endogenous alpha-peptide. The alpha-peptide from the pGEM®-Z Vectors or other lacZ containing plasmids complements the omega fragment of beta-galactosidase present in the bacteria. The resulting functional beta-galactosidase enzyme (alpha plus omega fragments) converts substrates such as X-Gal to a colored product, resulting in blue colonies. Cloning inserts into the multiple cloning region of the pGEM®-Z Vectors disrupts the alpha-peptide coding sequences, and thus inactivates the beta-galactosidase enzyme resulting in white colonies. Small inserts which happen to be in frame with the alpha-peptide coding region may produce light blue colonies, as beta-galactosidase activity is only partially inactivated. Recombinant plasmids are transformed into the appropriate strain of bacteria (i.e. JM109, DH5alpha), and subsequently plated on indicator plates containing 0.5 mM IPTG and 40 µg/ml X-gal. Alternately, 50µl of the X-Gal stock and 100µl of the IPTG stock can be directly added to each plate and the liquid dispersed over the entire plate and allowed to absorb into the agar for 30 minutes at 37°C. In general, IPTG is dissolved in sterile water to a stock concentration of 100mM, and X-Gal is dissolved in N,N'-dimethyl formamide (DMF) to a stock concentration of 50mg/ml. Both the IPTG and X-Gal should be aliquoted and stored at -20°C, and are stable for up to 2-4 months at this temperature.
satellite clonies represent resistance to your antibiotic it could happen after the needed time passes for bacteria to mutate and can growth insted of the Ab
In the case of amplicillin, Satellite colonies appears when selecting colonies resistant to Ampicillin, for example, resistance is mediated by a beta-lactamase which destroy the antibiotic: so when the colonies are big enough, the neighboring untransformed cells (they are alive because Ampi is a bacteriostatic, so, it doesn’t kill them) can form "micro" colonies around the transformed, but not instead of them.
What is the purpose of using IPTG?
If the LB plates contain kanamycin instead of amp,will i get satellite colonies?
1. why were there colonies and not lawns in the plate with Amp?
2. Are there any plasmid inserts in the white & blue colonies?
3. Does the plasmid disrupt the Lac Z gene in the cell?