This was given by the teacher during the experiment adn we have to compare it to

Question

This was given by the teacher during the experiment adn we have to compare it to the second picture of hte gel posted below. THE FIRST PICTURE IS A SAMPLE AND THE SCOND PICTURE IS WHAT WE OBTAINED IN THE LAB AND WRITE A DISCUSSION ON IT, ATKEAST ONE PAGE.

For mix reactions see methods page.1% ETBR @ 0.1 ?l/ml used to stain DNA in gel

1. Sample 1 in lane 1 is incubated at 0 oC without DNase

2. Sample 1 in lane 3 is incubated at 37 oC with Dnase

3. Sample in lane 4 is incubated at 37 oC without DNase.

4. Sample in lane 6 is incubated at 37 oC with DNase that has been pre heated to 90 C for 10 minutes.

5. Samples in lanes 7 & 8 are tRNA (10 ?l of **mg/ml), #7 with DNase & 8 no DNase both at 37 oC.

      Lanes 2 & 5 contains results of second prep not analyzed.

      Horizontal arrows indicate important species of nucleic acids (need to be labeled)

This is what we got in the lab during the experiment. Need to write a page of discussion to what was wrong with the result and what went wrong during the experiment that led to wrong results.

AGAIN, THE FIRST GEL IS GIVEN BY THE TEACHER AND THE SECOND GEL IS WHAT WE GOT IN THE LAB DURING THE EXPERIMENNT.

Explanation / Answer

Answer:

1.      Sample 1 in lane 1 of agarose gel that has been given by your teacher is a plasmid DNA preparation by alkaline lysis method. It was incubated at 0 deg without any DNAase. So you can see there are three different kinds of DNA bands. Those are linearized, supercoiled and circular DNA. Majority of the plasmid DNA will be in supercoiled form, so we call it as good prep as we need these DNA for cloning and transformation. Different forms of DNA shows up in agarose gel in terms of their relative migration speed. Circular single stranded plasmid DNA moves faster than supercoil, super coil moves faster than linearized DNA. The harse alkaline treatment gives permanently denatured single stranded DNA that migrate ahead of all forms of DNA.

2.In lane 3, sample 1 which was incubated with DNAase at 37 deg. Hence DNA becomes degraded/ cleaved, where as in lane 1 of sample 1 is not good prep DNA that stored at 0 deg

3.Sample in lane 4 is incubated at 37 deg without DNAase, so linearized DNA is very clear.

4.Sample in lane 6 is incubated at 37 with DNAase which was preheated, that means all the DNAase enzyme got deactivated , so enzymatic cleavage of DNAase . hence all different forms of DNA prep.

t-RNA which are of 76 to 90 nucleotide bases run in gel very faster than all different forms of prep DNA

In second ETBR gel, which was run by students, was not in right direction i.e the lane 1 becomes lane 8 here. So they just flip the gel from left to right. Another point is plasmid DNA were not quantitated properly. The amount of ETBR was added more. The alkaline lysis step was harse. So most of the DNA was single circularized form. These are not good plasmid DNA.

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