Assignement 2
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
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