Fledgling grad student, Joe Peon, is doing PCR and goes to the freezer to get th
ID: 90795 • Letter: F
Question
Fledgling grad student, Joe Peon, is doing PCR and goes to the freezer to get the reagents needed to set up the reaction. This overworked graduate student grabs dNTPs, 2 forward primers, enzyme buffer, and restriction enzyme Taql. out of storage and sets up his reaction tube. He sets up 2 tubes: 1) the positive control with target DNA added 2) the test sample + target DNA. He adds all the reagents listed, in the amounts given in the protocol and sets up the PCR cycler to cycle a heat phase for 30 seconds and extend for 1 minute. The next morning, he ran the samples out in an agarose gel. What, if anything will Joe Peon see on the gel? What mistakes were made (there are numerous errors - name 3 specific errors)? c. If you design a lysogenic bacteriophage to express a toxin, such as the diphtheria toxin, arid use this toxin bearing phage to infect E. coli, what would happen to the E.coli if this phage became a prophage? d. The leader sequence of the histidine operon has multiple histidine codons and the leader of the tryptophan operon has tryptophan codons. You place the histidine leader in front of the tryptophan structural genes and vice versa. If you grew the resulting cells in media with tryptophan but lacking histidine, would you expect histidine or tryptophan to be produced? Why or why not? e. You discover and describe an enzyme that works in the cytosol of E.coli to increase the stability of the mRNA of a structural gene. i. Would this affect the transcription or translation of a gene? ii. Would you expect more or less protein produced?Explanation / Answer
The master mix for PCR should be prepared carefully with the following ingradients:
Buffer,
dNTPs,
MgCl2 ,
Forward Primer,
Reverse primer,
Taq Polymerase and
RNase free water
Prepare a positive control and a negative control reaction mix, gently mix the reaction and spin down in microcentrifuge.
Set up the PCR as following:
initial denaturation for 30 seconds
denaturation, annealing and extension for about 30 cycles and
a final extension step.
Finallycarry out electrophoresis with the PCR end product on agarose gel.
Answer b1. Joe Peon can not see any thing on the agarose gel as he did multiple gross mistakes.
Answer b2. Some of the mistakes made by him are:
Mistake 1. Master mix was not prepared well. The master mix should contain the ingredients given above.
He forgot to add reverse primer in the reaction mixture. Instead he added two forward primers.
Mistake 2.He did not set up the reaction tubes properly.
while running a PCR reaction, 3 PCR tubes should be prepared,
i) A positive control
ii) Test DNA sample
iii) A negative control
A positive control is a PCR mix with DNA known to work in amplification + PCR master mix
A test DNA is a PCR mix with test DNA to be amplifed + PCR master mix
A negative control is water + PCR master mix
Mistake 3: He did not set up the PCR reaction cycles properly.
The PCR reaction should be set up with cycles as described above. (Initial denaturation; denaturation annealing extension cycles; and final extension)
His reaction has an initial denaturation and final extension steps. But the 3 critical steps of PCR (denaturation, annealing and extension cycles) were missing.
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