Please interpret each of the genetic Results A. PCR Agarose Gel Method for A. PCR reactions to be prepared 1 2 3 4 5 6 A
Posted: Thu Jun 09, 2022 1:44 pm
Please interpret each of the genetic Results
A. PCR Agarose Gel
Method for A.
PCR reactions to be
prepared
1
2
3
4
5
6
Annealing temperature
60oC X 5 cycles
63oC X 5 cycles
66oC X 5 cycles
56oC X 30 cycles
59oC X 30 cycles
62oC X 30 cycles
Genomic DNA (ng)
25
25
25
25
25
25
Primers
(final concentration for each primer in µM)
0.2
0.4
0.2
0.4
0.2
0.4
B. Western Blot
Method for B.
After transfer of proteins to nitrocellulose, laboratory staff
will incubate the membrane with one of the following antibodies: 1.
Rabbit monoclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)
2. Rabbit monoclonal anti-p44/42 MAPK (Erk1/2) The antibodies will
be diluted in a mixture of isotonic salt solution of 5% (w/v) of
skim milk powder and incubated overnight at 40C
C. gDNA Agarose gel
Method for C.
Preparation of Primer and gDNA
template
Reaction components
Stock concentrations
Final reaction volume for one PCR is
25µl
In this table you are
calculating volumes for 3.5 reactions
Master mix 1
Master mix 2
Final conc. or amount
µl to add
Final conc. or amount
µl to add
Primer
Concentration*
(µM)
2µM
0.2µM
10
0.4µM
20
Amount of gDNA
template (ng)
5ng/µl
25ng
5
25ng
5
OneTaq Mastermix
2X
1X
50
1X
50
Nuclease-free water
Add water such that the final total
reaction volume in each tube is 3.5 x 25µl
PLEASE LABEL WHICH RESULTS EXPLANATION A B AND C LIKE IN THE
IMAGES
500- 400- bp 1 2 3 4 5 6 7 8 9 10 11 12 1
--- OH O RAS phospho-ERK Total ERK MW A549 BEAS2B A549 BEAS2B A549 BEAS2B MW A549 BEAS2B A549 BEAS2B
3 L N BamHI ECORI HindIll BamHI ECORI HindIll BamHI ECORI HindIll Undigested gDNA Undigested gDNA N BamHI SECORI B Hindill BamHII ECORI HindIll Undigested gDNA 3 N BamHI ECORI HindIll BamHI I ECORI HindIll BamHI ECORI HindIll 6 R7
A. PCR Agarose Gel
- Please Interpret Each Of The Genetic Results A Pcr Agarose Gel Method For A Pcr Reactions To Be Prepared 1 2 3 4 5 6 A 1 (335.74 KiB) Viewed 210 times
PCR reactions to be
prepared
1
2
3
4
5
6
Annealing temperature
60oC X 5 cycles
63oC X 5 cycles
66oC X 5 cycles
56oC X 30 cycles
59oC X 30 cycles
62oC X 30 cycles
Genomic DNA (ng)
25
25
25
25
25
25
Primers
(final concentration for each primer in µM)
0.2
0.4
0.2
0.4
0.2
0.4
B. Western Blot
- Please Interpret Each Of The Genetic Results A Pcr Agarose Gel Method For A Pcr Reactions To Be Prepared 1 2 3 4 5 6 A 2 (300.71 KiB) Viewed 210 times
After transfer of proteins to nitrocellulose, laboratory staff
will incubate the membrane with one of the following antibodies: 1.
Rabbit monoclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)
2. Rabbit monoclonal anti-p44/42 MAPK (Erk1/2) The antibodies will
be diluted in a mixture of isotonic salt solution of 5% (w/v) of
skim milk powder and incubated overnight at 40C
C. gDNA Agarose gel
- Please Interpret Each Of The Genetic Results A Pcr Agarose Gel Method For A Pcr Reactions To Be Prepared 1 2 3 4 5 6 A 3 (718.96 KiB) Viewed 210 times
Preparation of Primer and gDNA
template
Reaction components
Stock concentrations
Final reaction volume for one PCR is
25µl
In this table you are
calculating volumes for 3.5 reactions
Master mix 1
Master mix 2
Final conc. or amount
µl to add
Final conc. or amount
µl to add
Primer
Concentration*
(µM)
2µM
0.2µM
10
0.4µM
20
Amount of gDNA
template (ng)
5ng/µl
25ng
5
25ng
5
OneTaq Mastermix
2X
1X
50
1X
50
Nuclease-free water
Add water such that the final total
reaction volume in each tube is 3.5 x 25µl
PLEASE LABEL WHICH RESULTS EXPLANATION A B AND C LIKE IN THE
IMAGES
500- 400- bp 1 2 3 4 5 6 7 8 9 10 11 12 1
--- OH O RAS phospho-ERK Total ERK MW A549 BEAS2B A549 BEAS2B A549 BEAS2B MW A549 BEAS2B A549 BEAS2B
3 L N BamHI ECORI HindIll BamHI ECORI HindIll BamHI ECORI HindIll Undigested gDNA Undigested gDNA N BamHI SECORI B Hindill BamHII ECORI HindIll Undigested gDNA 3 N BamHI ECORI HindIll BamHI I ECORI HindIll BamHI ECORI HindIll 6 R7