DNA in a gel is stained by ethidium bromide. The amount of ethidium bromide bound by a DNA molecule is proportional to the length of that DNA molecule. Therefore, long molecules will fluoresce more brightly than short molecules.
All engineered plasmids have three components: 1. An origin of replication (to replicate the plasmid in its host cell) 2. A gene encoding a selectable marker (i.e. ampicillan resistance) 3. Multiple restriction enzyme digestion sites (for subcloning fragments of DNA into that plasmid)
Forms which plasmid DNA can occur in: 1. Supercoiled – condensed, tightly packed DNA 2. Nicked – where there is a DNA break on one strand that relaxes the supercoiling of the DNA. 3. Linear – occurs once the plasmid DNA has been digested with a restriction enzyme, creating a double-stranded break in the DNA.
The linear form of the DNA shows the TRUE molecular size.
The ladder we use to determine approximate sizes of DNA fragments is bacteriophage λ DNA cut with HindIII (concentration = 100 ng/μl). We usually load 12 μl into the well.
|Plasmid # |Description |Details |
|1 |pBluescript KS+ |General subcloning/sequencing plasmid |
|2 |pBluescript-like vector + cDNA1 |cDNA1 into pBS |
|3 |pBluescript-like vector + cDNA2 |cDNA2 into pBS |
|4 |pBluescript-like vector + cDNA3 |cDNA3 into pBS |
|5 |Taq plasmid #33 |Taq polymerase encoding plasmid |
|6 |pGBT9 |Yeast protein expression plasmid |
|7 |pACT-2 |Yeast protein expression plasmid |
We use agarose which is a 1% w/v solution kept at 60˚C. A lower concentration of agarose is used for separating larger DNA fragments and higher concentrations for separating smaller DNA fragments.
We use 1X TAE buffer when we run our gels: 1. Tris base 2. glacial acetic acid 3. EDTA, pH 8.0
We use blue juice as our sample buffer, it has two main components: 1. Sucrose – to weigh down the sample so it will not float out of the well 2. Bromophenol Blue – a tracking dye for migration of the sample through the gel
We usually load 0.5 μg of DNA. The composition of one well usually looks like: 1. 2 μl of blue juice 2. 16 μl of water 3. 2 μl of DNA (this can vary depending on the concentration of the DNA) = 20 μl TOTAL
As you know, we have had problems with gels melting. So now, we don’t run the gels above 80V. We run the gels so the blue marker runs ½ to ⅔ the length of the gel. Remember, RUN TO RED.
Restriction Enzymes used in Lab: 1. EcoRI 5’NNG AATTCNN3’ 3’NNCTTAA TTAAGNN5’
2. BamHI 5’ NNG GATCCNN3’ 3’NNCCTAG GNN5’
Both of these enzymes provide a 5’- overhang.
Transformation – the introduction of DNA into yeast or bacteria
Transfection – the introduction of DNA into insect or vertebrate cells growing in a tissue culture.
When performing a digestion, we usually use the following protocol:
1 μl of DNA (can be greater)
2 μl of 10X restriction enzyme buffer
16 μl of DI water
1 μl of restriction enzyme (added last)
= 20 μl TOTAL
When transforming E.coli cells, we need them to be in the exponential stage of their growth. This occurs at an OD600 = 0.2 to 0.6 of a 1:100 diluted 5 mL LB. (~3 hrs).
1 OD600 ≈ 8.8∙108 cells/ml
After transforming the E.coli cells we need to verify that the correct plasmid was inserted. The