Biology homework help

| December 24, 2015

DNA practical Write-up

BIT002 Practical Project

Introduction: This project is based on the methods for analyzing human stem cell mutants generated by CRISPR. We have created a series of hESC lines, generated from a CRISPR-cas9 knockout construct that targets the NMDA receptor subunit, GRIN2A. In this project, you will analyse the DNA made from 3 hESC lines, looking for evidence of mutations in the GRIN2A gene.

 

As the cell possesses two copies of each gene (2 alleles), there are three possible outcomes of the mutagenesis in each cell:

 

  1. i) no mutation is incorporated, therefore all alleles are wild type;
  2. ii) only one allele is mutated, therefore the cell line is heterozygous;

iii)   both alleles are mutant, therefore the cell line is homozygous.

 

Can you identify, the nature of the mutations in each cell line to be tested (1,2 & 3)?

 

In this practical exercise, we will carry out 3 experimental procedures.

 

  1. PCR analysis of genomic DNA for each cell line;
  2. Cloning the PCR fragments for sequencing;
  3. Sequence analysis of the resultant clones.

 

This will involve both a 3 hour lab based practical and a computer based analysis.

 

Write-up should include a full description of the Results and a Discussion that includes: what you have learnt about your CRISPR mutants; what you would do next with your mutants and what improvements you could to make the screening process more efficient and faster.

 

 

Experimental Procedures:

 

Experiment 1: PCR analysis

 

First you will investigate 10 ml of each PCR product (provided ready for electrophoresis) using agarose gel electrophoresis.

 

 

  1. Set up gel tray with rubber end pieces to seal the ends and a comb place close to one end.
  2. Pour in 50mls of 2% agarose/dye held at 55°C and allow to solidify.
  3. When set, remove end pieces and place in gel box and cover with buffer and Remove comb from submerged gel.
  4. Load 10µl of Marker (= 1Kb DNA Ladder), then 10 ml of each of the 3 PCR products

         M       PCR PRODUCT 1       PCR PRODUCT 2       PCR PRODUCT 3

  1. Run gel for 1-1.5 hours, until Bromophenol dye reaches the bottom.
  2. Visualise and photograph gel on a trans-illuminator.

Experiment 2: TOPO cloning of PCR products

PCR products can be readily cloned into TOPO vectors. The aim of this experiment is to clone a PCR product (tube P) into a TOPO vector, and transform E. coli.

 

  1. Set up the following reactions
  2. i) 1µl of TOPO vector (tube T) alone
  3. ii) 1µl of TOPO vector plus 5µl of PCR product (tube P).

Make each tube up to 10µl with H2O and incubate on ice for 5 minutes

  1. Thaw out a tube of transformation competent cells, and add 25µl to 2µl of each reaction and incubate on ice for 30 minutes
  2. Heat shock the cells for 30 seconds exactly, then return to ice for 5 mins.
  3. Add 200µl of SOC medium to each tube and incubate at 37°C for 60 minutes

 

  1. Label the 2 plates (not the lids) with your initials and whether it was tube T or P.

 

  1. Incubate, inverted overnight at 37°C. (plates will be stored at 4°C ).

 

  1. Identify your plates and look at what has or hasn’t grown.

Experiment 3: Analysis of mutant clone sequences. (to follow)

1 Kb DNA Ladder

A number of proprietary plasmids are digested to completion with appropriate restriction enzymes to yield 10 bands suitable for use as molecular weight standards for agarose gel electrophoresis. The digested DNA includes fragments ranging from 0.5-10.0 kilobases (kb). The 3.0 kb fragment has increased intensity to serve as a reference band. The approximate mass of DNA in each of the bands is provided (assuming a 0.5 μg load) for approximating the mass of DNA in comparably intense samples of similar size.

0.5 µg /lane

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