Question

In: Biology

•In colony PCR of E. Coli, we were amplifying the GFP gene. In restriction digest, we...

•In colony PCR of E. Coli, we were amplifying the GFP gene. In restriction digest, we were cutting out the GFP gene. The GFP gene is about 700 bp (base-pairs) and the entire plasmid including the GFP gene is about 3700 bp. Either draw out or explain where you would expect to see bands on the gel for each sample:
•a) single digest, b) double digest, c) uncut miniprep, d) GFP PCR, e) control PCR

Solutions

Expert Solution

Ans:- Single Digest

For single digest of the plasmid containing GFP gene will give a linear band of 3700 bp. Because, GFP cloned into plasmid is 3700 bp and upon single digestion restriction enzyme will cut the plasmid at single site. If circular plasmid is cut once it will become linear and size of the plasmid remain the same.

Double Digestion

Since the size of GFP is 700 bp. If you double digest the plasmid with enzymes it will gives two linear bands one of 700 bp for GFP and other is 3000 bp the plasmid backbone. The logic is that the GFP is cloned using these two restriction site only into the plasmid, so if you digest plasmid with these two enzymes the GFP band of 700 bp will come out from plasmid and 3000 bp of linear backbone is also present.

Uncut plasmid

The uncut plasmid is the supercoiled plasmid purified from the mediprep. So, when uncut plasmid is run on the gel the actual size of plasmid is bite difficult to predict. So, the supercoiled plasmid will give you a band anything from 3000 bp to 3700 bp. As, supercoiled plasmid can move faster than linear plasmid.

GFP PCR

Since, the GFP is of 700 bp. Thus, the GFP PCR will produce a linear band of 700 bp.Since, primers will only amplify the size of the GFP.

The control PCR

The control PCR won't give any band of GFP because it doesn't contain GFP gene. So, no band will be seen in control PCR.

Dear student if you find the answer convincing please do give the feedback.


Related Solutions

What will you mix together in your restriction enzyme digest to cut out the gfp gene?
What will you mix together in your restriction enzyme digest to cut out the gfp gene?
Consider a colony of E. coli cells in which the gene for the CRP protein is...
Consider a colony of E. coli cells in which the gene for the CRP protein is deleted. Describe the effect of that this deletion will have on the arabinose operon in terms of levels of Ara A protein (one of the proteins encoded by a gene in the ara operon) when the cells are grown in media containing arabinose but no glucose. Explain your reasoning.
Consider a colony of E. coli cells that have a mutation in the lac I gene,...
Consider a colony of E. coli cells that have a mutation in the lac I gene, resulting in a LacR protein that cannot bind to allolactose. Describe the effect that this mutation will have on the lac operon in terms of βgal protein levels when the cells are grown in media containing lactose but no glucose. Explain your reasoning.
The E. coli LacZ will be used as a reporter gene and the expression of the...
The E. coli LacZ will be used as a reporter gene and the expression of the LacZ gene product will be detected by the enzymatic cleavage of the compound X-gal producing blue staining within the embryo. Why was an E. coli LacZ gene used as a reporter gene? Why is there a Drosophila weak basal promoter located on the P[lacZ] enhancer-trap element? The E. coli LacZ will be used as a reporter gene and the expression of the LacZ gene...
Describe the colony characteristics of E. coli, Bacillus cereus, Bacillus subtilis, Staphylococcus epidermidis
Describe the colony characteristics of E. coli, Bacillus cereus, Bacillus subtilis, Staphylococcus epidermidis
During “cut and paste” cloning, the PCR-amplified gene is cut by restriction enzymes. How does the...
During “cut and paste” cloning, the PCR-amplified gene is cut by restriction enzymes. How does the synthesis of DNA oligonucleotide primers facilitate the ability to ‘cut’ the amplified gene?
Assume that the gene trpA in an auxotrophic strain of E. coli is located at 27...
Assume that the gene trpA in an auxotrophic strain of E. coli is located at 27 minutes, whereas the gene pyrE is located at 81 minutes. Present an experimental scheme that would allow you to convert one of the auxotrophic strains to a prototrophic strain. A. F+ (auxotroph) × F− (wild type) B. Hfr (auxotroph) × F− (auxotroph)   C. Hfr (auxotroph) × F− (wild type) D. Hfr (wild type) × F− (auxotroph) E. F+ (wild type) × F− (auxotroph) The...
The RY13 strain of the bacteria E. coli makes the restriction enzyme EcoR I, which cuts...
The RY13 strain of the bacteria E. coli makes the restriction enzyme EcoR I, which cuts at the sequence GAATTC. this occurs thousands of time in a full chromosome. Why doesn’t the enzyme cut the chromosome into tiny bits, killing the bacterium?
Your lab is studying gene expression of the tryptophan operon in the bacterium E. coli. In...
Your lab is studying gene expression of the tryptophan operon in the bacterium E. coli. In order to facilitate this study, you have cloned the Trp-operon, excised the five coding genes (TrpA, B, C, D, and E), and replaced them with a copy of the reporter gene GFP (green fluorescent protein). The resulting recombinant DNA was inserted into an F' plasmid, and used to transform a number of different strains of E. coli. For each of the scenarios listed below,...
Provide detailed descriptions of the events of gene translation. Use E. coli as your model and...
Provide detailed descriptions of the events of gene translation. Use E. coli as your model and include i.) initiation, ii.) elongation, and iii.) termination.
ADVERTISEMENT
ADVERTISEMENT
ADVERTISEMENT