- Explain how you were able to identify plasmid in each sample using the gel, considering how linear DNA fragments of different length are separated by agarose gel electrophoresis.- Why do supercoiled, nicked and linear DNA sequences of the same size (kb) migrate at different rates during agarose gel electrophoresis?- How does SYBR Safe® enable the visualisation of the location of DNA fragments on the gel? Materials• Plasmid DNA sample at a concentration of 100 ng/uL in sterile, nuclease-free water, labelled "P"•ECOR1 restriction enzyme10x concentrated restriction enzyme buffer• Nuclease-free waterDraw a simple diagram of the four plasmids showing the EcoRI recognition site(s). Think about thelinear DNA products with staggered (sticky) ends that will be produced, assuming digestion is fullysuccessful.11 of 14ECOR1 digestsPUC-4KpBC4KpBC-SK-PUC-18PART TWO: AGAROSE GEL ELECTROPHORESIS OF PLASMID DNA SAMPLESMaterials6x agarose gel loading dyeSterile distilled water (dH₂O)TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0, 1x SYBR Safe® dye)Pre-prepared 0.8% agarose gel (8 g/L agarose in TBE buffer) containing SYBR Safedye1 kb marker ladder of linear DNA fragments (1 to 12 kb)MethodLane 1 Lane 2DNA Plasmid 1ladder "ND"Lane 3Plasmid 1"D"Model DataLane 4Plasmid 2"ND"#3Lane 5Plasmid 2"D"1. Confirm that the sample from above has not evaporated. If the sample has evaporated significantly, itcan be made up to 10 μL with dH₂O using a P20 pipette, gently mixing for 5 min to ensure that any DNAdried onto the tube has dissolved. Prepare Part 2 samples by adding 1.6 μL only of 6x agarose gel loadingdye to both ND and D. Centrifuge for 10 s to ensure all liquid is at the bottom of the tube.#22. The gels and buffer contain SYBR Safe dye - a less mutagenic and safer alternative to ethidiumbromide. Take care to dispose of P200 tips into the biohazard bags immediately after use. There will be ademonstration of how to load the gel with 1 µL the 1 kb marker ladder sample. Load 8 μL of the "D" and"ND" samples into adjacent sample wells in the gel in the order ND, D.Lane 6Plasmid 3"ND"#1-10.0 42-8.0-6.0 50-5.0-40 33#442ND DND DND D ND42-3.0 125Lane 7 Lane 8Plasmid 3 Plasmid 4"D" "ND"-2.0 48-1.5 36-1.0 42-0.5 42Lane 9Plasmid 4"D"

Agarose gel electrophoresis is a commonly used technique to separate DNA fragments based on their…

Answered: -3.0 3.0 125 -20 48 -15 36 -1.0 42 -05…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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- Explain how you were able to identify plasmid in each sample using the gel, considering how linear DNA fragments of different length are separated by agarose gel electrophoresis.
- Why do supercoiled, nicked and linear DNA sequences of the same size (kb) migrate at different rates during agarose gel electrophoresis?
- How does SYBR Safe® enable the visualisation of the location of DNA fragments on the gel?

Materials
• Plasmid DNA sample at a concentration of 100 ng/uL in sterile, nuclease-free water, labelled "P"
•ECOR1 restriction enzyme
10x concentrated restriction enzyme buffer
• Nuclease-free water
Draw a simple diagram of the four plasmids showing the EcoRI recognition site(s). Think about the
linear DNA products with staggered (sticky) ends that will be produced, assuming digestion is fully
successful.
11 of 14
ECOR1 digests
PUC-4K
pBC4K
pBC-SK
-
PUC-18
PART TWO: AGAROSE GEL ELECTROPHORESIS OF PLASMID DNA SAMPLES
Materials
6x agarose gel loading dye
Sterile distilled water (dH₂O)
TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0, 1x SYBR Safe® dye)
Pre-prepared 0.8% agarose gel (8 g/L agarose in TBE buffer) containing SYBR Safe
dye
1 kb marker ladder of linear DNA fragments (1 to 12 kb)
Method
Lane 1 Lane 2
DNA Plasmid 1
ladder "ND"
Lane 3
Plasmid 1
"D"
Model Data
Lane 4
Plasmid 2
"ND"
#3
Lane 5
Plasmid 2
"D"
1. Confirm that the sample from above has not evaporated. If the sample has evaporated significantly, it
can be made up to 10 μL with dH₂O using a P20 pipette, gently mixing for 5 min to ensure that any DNA
dried onto the tube has dissolved. Prepare Part 2 samples by adding 1.6 μL only of 6x agarose gel loading
dye to both ND and D. Centrifuge for 10 s to ensure all liquid is at the bottom of the tube.
#2
2. The gels and buffer contain SYBR Safe dye - a less mutagenic and safer alternative to ethidium
bromide. Take care to dispose of P200 tips into the biohazard bags immediately after use. There will be a
demonstration of how to load the gel with 1 µL the 1 kb marker ladder sample. Load 8 μL of the "D" and
"ND" samples into adjacent sample wells in the gel in the order ND, D.
Lane 6
Plasmid 3
"ND"
#1
-10.0 42
-8.0
-6.0 50
-5.0
-40 33
#4
42
ND DND DND D ND
42
-3.0 125
Lane 7 Lane 8
Plasmid 3 Plasmid 4
"D" "ND"
-2.0 48
-1.5 36
-1.0 42
-0.5 42
Lane 9
Plasmid 4
"D"

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What legend would you give to the labeled model data?

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Looking at the model data i upload and given the question how could i analyse tboth experimental results (antibiotic selection and agarose gel). Including one clearly labelled image of the agarose gel and with an accompanying legend. And how could i interpret the findings of the results and draw a conclusion i.e. what is in each tube? Also is there any further experiments that I could perform to confirm the identity of the plasmid stocks?

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