LAB - pGLO Restriction Digest

pGLO plasmid map
click on picture for a full size image

Following the plasmid isolation procedure using minipreps, our next step would be the one we would follow if we were cloning a gene: Isolate the gene from the plasmid. In this case we want to isolate the GFP gene, and we will be using the BamH I restriction enzyme, which cuts GFP from the plasmid, and it also cuts the gene in two fragments.

First we ran an agarose gel to know if we actually have succesfully isolated our plasmid DNA (we do know we have plasmid DNA in some of our section 2 samples, but it is still unclear in our section 1 samples). After that, we set up the restriction enzyme digest (RED).

Next week we will run a polyacrylamyde gel (PAGE) that we will silver stain. Silver staining is a very sensitive technique to visualize proteins and DNA in agarose and polyacrylamide gels.

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LAB - Plasmid isolation using mini-preps

We have completed a cycle, and after genetically tranforming bacteria using Bio-Rad's pGLO pasmid, it's time to isolate it from our clones. We used the Wizard® Plus Minipreps DNA Purification System (Promega), which provides a simple and reliable method for rapidly isolating plasmid DNA. This system can be used to isolate any plasmid but works most efficiently when the plasmid is <20,000bp. Click here to view a pdf of the complete protocol of the miniprep kit.

We lysed the bacteria and added a series of reagents to isolate plasmid DNA wothout getting any bacterial genomic DNA. The ultimate objective is to isolate the GFP gene from the rest of the plasmid.

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Control of gene expression

Different levels at which gene control can be exerted
(click on pic for full size image)

We started considering the mechanisms through which cell "knows" what subset of the genetic information must express in order to become the cell that it's supposed to be (a neuron, a lymphocite, a pancreatic cell, etc...). We considered the different levels at which a cell can control what proteins and RNAs are produced.

We also introduced the concept of gene regulatory proteins (a.k.a. transcription factors) and their relation to regulatory DNA sequences., and how they can read the information coded in the DNA without openeing the double helix, based solely on unambiguous chemical properties of the phosphate backbone of each base pair in the major grooves of the helix.

Material covered: pages 411-418

Reading for next class: pages 418-454

Quiz 12 Qs&As:

1. What is DNA repair?
The mechanism to correct changes in DNA sequence after transcription (after proofreading), before the information is passed to daughter cells. It is performed by a battery of enzymes, and not by a single one.

2. Mention or explain (briefly!) a way in which DNA damage can be removed
Base excision repair, nucleotide excision repair

3. What happens to DNA sequence when it is repaired by non-homologous end joining compared to when it is repaired by homologous recombination?
In non-homologous end joining one or more nucleotides of each strand are deleted.
In homologous recombination the information of the missing piece of strand can be copied from the sister chromatid and restored.

4. Which protein detects DNA damage during the transcription process?
RNA polymerase

5. What is control of gene expression?
Biochemical mechanisms to express selected genes and repress those that are not necesary

DNA Repair

Today we had an activity which goal is to improve class participation (and preparation). After doing a quick review of last class, based on the replication "fork" figure (see entry on Wednesday October 22nd - 'DNA replication machinery'), students prepared a 1-2 page essay on today's topic: DNA repair. Students were allowed (and encouraged) to consult the textbook, discuss topics, and ask questions.

The essay will be worth 10 points (to be added to the quiz scores)

Material covered can be found in pages 295-304. Main topics to consider for the exam are:

• Importance of DNA repair
• Repair of the double helix
• Pathways of DNA repair
• DNA polymerases specialized on DNA repair
  
• Repair of double strand breaks
• Effect of DNA repair on the cell cycle

Reading for tomorrow: Control of gene expression (p 411-454... don't freak out. Not every single word will be covered. But read the whole thing if possible)

Quiz #11 Q&As

1. What is a replication fork?
Enzyme induuced 'y' shape conformation of the DNA replicaiton helix

2. What does DNA polymerase do?
It synthesizes mRNAs based on molecualr infromtion

3. What does DNA primase do?
It binds a small RNA promer to the template strand of DNA so the polymerase can start extending the sequence

4. What is DNA repair?
Mechanisms to correct post-proofreading DNA changes, performed by a variety of enzymes

5. How are DNA repair and proofreading different?
Proofreading is done only by some polymerases. DNA repair involves many enzymes

6. What are Okazaki fragments? (bonus: 3 points)
During DNA replication the short fragments of DNA resulting from replication of the lagging strand taking place in the opposite direction to that in which the replication fork opens. They are later stuck together by a DNA ligase to form a continuous strand

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