David Quinn
pGLO Transformation
Lab
Procedure in past passive
By David Quinn
1. One of the two micro test tubes was labeled +pGLO and the
other micro test tube was labeled –pGLO. The two test tubes were then placed in
the foam tube rack.
2.250 micro-liters of CaCl2 was transferred to each test
tube using a sterile transfer pipet.
3.The foam tube rack was then transferred to an ice bath.
4.Roughly 2-4 large bacteria colonies were removed from the
starter plate using a sterile loop. The sterile loop was then placed into the
bottom of the +pGLO tube and spun around until all of the bacteria colonies
were dispersed into the solution. The test tube was then placed back into the
tube rack in the ice bath. The steps for the +pGLO test tube were then repeated
for the –pGLO test tube.
5.The pGLO DNA solution was observed under a UV lamp. A new sterile loop was then immersed into the
pGLO plasmid DNA stock tube. A loop-full
was removed and added to the +pGLO tube. Both test tubes were then closed and
returned to the ice bath.
6.The test tubes were pushed all the way through the tube
rack so that the bottom of the test tube makes contact with the ice. The test tube rack was left in the ice for 10
minutes.
7.The four LB nutrient agar plates were then labeled on the
bottom. One LB/amp plate was labeled
+pGLO, one LB/amp/ara plate was labeled +pGLO, the other LB/amp plate was
labeled –pGLO and the LB plate was labeled –pGLO.
8 .The test tube rack was then removed from the ice bath and
transferred to another water bath set at 42 degrees celsius for 50 seconds. The
temperature of the water bath was double-checked with two thermometers to
ensure accuracy. After the 50 seconds
was over, the test tubes were immediately put in the ice bath for another 2
minutes.
9.The test tube rack was removed and placed on the bench.
250 micro-liters of LB nutrient broth was added to each test tube using a new
sterile pipet. The test tubes were then incubated at room temperature for 10
minutes.
10. The test tubes were flicked and mixed to re-suspend the
bacteria. 100 micro-liters of the transformation and control suspensions was
added to the appropriate nutrient agar plates using a new sterile pipette.
11.A new sterile loop was used for each plate to evenly
spread the suspensions around the surface of the LB nutrient agar by skating
the flat surface of a new sterile loop back and forth across the plate surface.
12. The plates were then stack upside-down and taped
together. The stack was then labeled and placed in an incubator at 37 degrees
celsius until the next day.
Lesson 2 review questions
By David Quinn
1.You would expect to find bacteria most like the original
non-transformed E.coli colonies initially observed in the LB –pGLO plate
because this is the control plate. No
plasmid was added to this plate so there will be no glow when under a UV light.
2.The plate that will be most likely to have genetically
transformed bacterial cells
will be the +pGLO LB/amp/ara plate because it contains the
gene that glows under the UV light and is resistant to ampicillin so it
contains the most likely bacteria colonies to survive.
3.The +pGLO LB/amp/ara plate should be compared to the –pGLO
LB plate because the LB plate will not glow under the UV light so you can
accurately determine if the LB/amp/ara is actually glowing or not.
4.A control plate is a plate that goes under the same
conditions as the other bacteria and has a known result. The purpose of a
control is to prove the results of the bacteria is reliable and that there are
no issues with the test itself.
