Assignement 2

Topics: Vibrio fischeri, Molecular biology, Plasmid Pages: 8 (905 words) Published: November 12, 2014
Genomic Library
A genomic library is a collection of recombinant vectors that represent the entire genome of an organism. Below is a brief outline of how to create the genomic library: 1. We isolated Vibrio fischeri DNA to pGEM genomic DNA using a phenol chloroform purification method. 2. Vibrio fischeri DNA and pGEM DNA was cut with restriction enzyme Sal I to prepare both for ligation. 3. Vibrio fischeri and pGEM DNA were ligated with T4 ligase. 4. E.coli DH5α competent cells were prepared for us so that transformation of the recombinant plasmids and the competent cells through heat shock transformation. 5. The transformation was then plated on LB/amp/X-gal plates. 6. The plates were screened for our target with ampicillin resistance, blue/white colony, then bioluminescence. 7. A bioluminescent was selected and re-streaked.

8. A non-glowing white colony was selected sent out for sequencing.

Ligation

a.
Tube
Insert to Vector Ratio
Digested V. fischeri (insert) DNA
Digested pGEM
H2O
5X Ligation Buffer
Total Volume
L1
2:1
0.30 µg = 1.5 µl
0.05 µg = 2 µl
20.5 µl
6 µl
30 µl
L2
3:1
0.45 µg = 2.25 µl
0.05 µg = 2 µl
19.75 µl
6 µl
30 µl
L3
4:1
0.60 µg = 3 µl
0.05 µg = 2 µl
19 µl
6 µl
30 µl
L4
5:1
0.75 µg = 3.75 µl
0.05 µg = 2 µl
18.25 µl
6 µl
30 µl
L5
____
____
0.05 µg = 2 µl
22 µl
6 µl
30 µl

The varied ratio of Vibrio fischeri DNA to pGEM DNA was varied with each tube to optimize the desired ligation product. We are trying to isolate the gene that encodes for bioluminescence. By varying the ratio, you have a higher chance of the target gene.

b.
Lane 1: HIND III standard
Lane 2: L1/T0
Lane 3: L1/Tend
Lane 4: L2/T0
Lane 5: L2/Tend
Lane 6: L3/T0
Lane 7: L3/Tend
Lane 8: L4/T0
Lane 9: L4/Tend
Lane 10: L5/T0
Lane 11: L5/Tend

c. The gel appeared to be in good condition with no breaks or tears. The gel did not seem to be punctured thus we were able to analyze the gel. Based on the gel, it can be seen that the ligation did not work. The Tend samples for the different ligations did not appear in any of the lanes. Furthermore, at the T0 samples we should have seen religated pGEM, the most common product. Had the ligation worked, comparing the T0 and Tend, the linear pGEM, which runs around 3200 bp relative to the Hind standard, would have either disappeared or faded in the Tend samples because an insert would have entered the plasmid making the size different. Since no bands showed up in Tend and there were no linear pGEM in the T0 samples, it was concluded that the ligation did not work.

Transformation
a. The negative control was the TE buffer. The positive control was the pGEM. The negative control tells us that colonies do not grow without the presence of an ampicillin resistant vector. The positive control tells us that colonies do grow if they contain ampicillin resistance. The controls worked as expected because for the negative control no colonies were observed while a few colonies were observed in the positive control. b. Based on T6 plates, the transformation efficiency was: 3.0 x 104 No colonies were observed for 10µl.

100 µl:

Had there been colonies observed in the plate with 10µl, the transformation would have been the same because it comes from the same stock solution. c.
d.
e. The results indicate that the transformation was successful. In each transformation, there was presence of colonies meaning that the colonies were ampicillin resistant. This could either mean that the pGEM relegated with itself or that the vector was inserted into the plasmid. The blue/white screening indicated whether the colonies were either ligated pGEM or that the transformation occurred. The blue colonies were the religated pGEM because if the vector was inserted into the plasmid, it would disrupt the lacZ gene making the colony unable to process X-gal which would not make it blue but instead white. Since...
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