genetics lab practical

1,000 mL = ?L

1L

1 mL = ?L

0.001 (10-3) L

1 mL = ? μL

1,000 μL

1 μL = ? L

0.000001 (10-6) L

step 1 for pipetting

you will first set the dial to the appropriate volume

step 2 for pipetting

next, you will place a plastic pipet tip onto the end of the pipetman (this will hold your solution)

step 3 for pipetting

you will slowly depress the knob at the top of the pipetman until it stops

step 4 for pipetting

you will place the tip into thesolution and gently release the pipetman knob to draw up the liquid

step 5 for pipetting

you will place the pipet tip, containing liquid, into a new tube and depress the pipetman knob all the way to release the liquid

pipetman first stop

you push the pipetman knob down to this stop only and place the pipet tip gently into your sample and release the knob slowly in order to draw up the sample

pipetman second stop

To release the liquid in the pipet tip, press the knob down all the way to this stop to ensure that all liquid has been expelled from the tip, and keep pressing the knob down as you remove the tip from the tube or solution (so you don’t suck any liquid back into the tip)

The polymerase chain reaction (PCR)

is a technique for replicating DNA in a tube that results in the exponential amplification of a selected region of a DNA molecule. This technique allows us to make billions of copies of any gene or sequence we are interested in working with, and because PCR only requires a miniscule amount of starting material, it can be used to obtain sequences from trace amounts of DNA present in material left at crime scenes, such as hair or bloodstains, or from bones discovered from murder victims or those found at archaeological sites.

PCR requires several basic components. These are:

1) DNA Template, the starting material which will contain the target region of DNA that we wish to amplify

2) Primers, which are needed to initiate DNA synthesis by the enzyme DNA polymerase, and which also determine the ends of the DNA fragment to be amplified.

3) Taq polymerase, a DNA polymerase isolated from the bacteria Thermus aquaticus, which lives in hot springs (between 95-100º C). It is used to synthesize many copies of the target DNA.

4) Deoxyribonucleotide triphosphates (or dNTP’s), the DNA monomers that the polymerase uses to build new DNA polymers.

5) Buffer solution, which maintains a stable and favorable salt concentration and pH for the Taq polymerase enzyme.

6) Divalent cations often act as enzyme cofactors and MgCl2 is required for the activity of strand extension by Taq polymerase (usually MgCl2 at 1-2.5 mM).

7) Double-distilled H20 (ddH2O), extremely clean water that has been autoclaved, so it is both free of impurities and sterile, which is added to the PCR to bring all components to their final working concentrations

DNA Template

the starting material which will contain the target region of DNA that we wish to amplify

Primers

which are needed to initiate DNA synthesis by the enzyme DNA polymerase,

and which also determine the ends of the DNA fragment to be amplified

Taq polymerase

a DNA polymerase isolated from the bacteria Thermus aquaticus, which lives in hot springs (between 95-100º C). It is used to synthesize many copies of the target DNA

Deoxyribonucleotide triphosphates (or dNTP’s)

the DNA monomers that the polymerase uses to build new DNA polymers

Buffer solution

which maintains a stable and favorable salt concentration and pH for the Taq polymerase enzyme

Divalent cations

often act as enzyme cofactors and MgCl2 is required for the activity of strand extension by Taq polymerase (usually MgCl2 at 1-2.5 mM)

Double-distilled H20 (ddH2O)

extremely clean water that has been autoclaved, so it is both free of impurities and sterile, which is added to the PCR to bring all components to their final working concentrations

why are primers are used in PCR

to direct the DNA polymerase to the designated piece of target DNA that is to be amplified. Primers are needed because DNA polymerases cannot begin synthesizing new DNA polymers from scratch, but they can add monomers to the 3’ end of an existing, single-stranded DNA polymer

where is the the PCR process carried out

in a thermal cycler

what does a thermal cycler do

This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction.

A typical PCR program is as follows:

94º C for 3 minutes

30-40 cycles of:

94º C for 30 secs

56-64º C for 30 secs

72º C for 1-3 minutes

Followed by:

72º C for 5 minutes

10º C indefinitely

denature PCR step

The 94º C steps are used to denature our double stranded DNA template into single strands.

pcr anneal step

The temperature is then lowered to allow the primers to anneal to the template strands – the single-stranded primers will form complementary base pairs with the template DNA.

pcr elongation step

The temperature used in this step will depend on the sequence of the primers used. Taq polymerase (or Pfu polymerase) elongates the new strand efficiently at a higher temperature, like 70-72º C, so we have to increase the temperature to optimize DNA synthesis, and the amount of time spent in this step depends on the size of the piece of DNA we want to copy.

Plasmids

double-stranded circular DNA molecules that are replicated in bacteria, and if the plasmid is called an expression vector, it contains embedded promoters that allow for transcription of recombinant genes (expression)

Most plasmids have three important components:

an antibiotic resistant marker, polylinker (also called a multiple cloning site or MCS), and an origin of replication (oriC; origin of chromosomal replication)

plasmid map

Because DNA sequencing technology has advanced over the years, scientists now have the complete DNA sequence of plasmids. These sequences can be used with computer programs to identify restriction sites on the DNA, as well as other features

origin of replication

contains the information to facilitate plasmids to replicate (DNA synthesis or replication) autonomously from the host genome (chromosome) of bacteria.

lab 2 goal:

to change a kanamycin resistant marker (gene) to an ampicillin resistant marker on a plasmid

what’s vehicles

its some of the most elaborate plasmids that contain promoters (DNA switches) that allow for expression of foreign genes that are inserted into MCS’ of plasmids and are denoted as prokaryotic expression plasmids

MCS represents

a cluster of unique restriction sites within plasmids that allow an investigator the ability to open up the plasmid (restriction digestion) and insert foreign pieces of dsDNA at this location, while the origin of replication contains the information to facilitate plasmids to replicate (DNA synthesis or replication) autonomously from the host genome (chromosome) of bacteria

how to replicate or express a gene on a plasmid

it first must be delivered into bacteria by the process of transformation

a selectable marker

allows scientists to rapidly find bacteria that pick up plasmids, while getting rid of bacteria that do not uptake plasmids (non-transformed parental bacteria) due to this poor transformation efficiency

In order to accomplish changing the kanamycin selectable marker on the pET24a MaSp1 4x vector to an ampicillin selectable maker, we will need to perform the following steps:

1) We will need to use polymerase chain reaction (PCR) to amplify the AmpR gene from pBluescript II SK+, a different plasmid that serves as a template source for the AmpR gene (Fig. 2) and;

2) We will need to isolate the prokaryotic expression vector pET24a MaSp1 4x, which will be used to insert the amplified PCR product (AmpR gene)

how to isolate plasmid from other bacterial components and how to retrieve plasmid DNA

you will need to break open the cells and use a purification method to separate your pET24a MaSp1 4x cloning vector from the host genome, RNA, protein and other bacterial macromolecules. technique: alkaline lysis and then adsorption of DNA onto a silica resin in the presence of high salt

The procedure consists of three basic steps: (lab 2)

• Preparation and clearing of the bacterial lysate

• Adsorption of DNA onto a QIAprep membrane

• Washing and elution of plasmid DNA

step 1 of Plasmid DNA isolation (lab 2)

Transfer 1.5 ml of the liquid culture into an eppendorf tube and centrifuge the sample in a microcentrifuge for 5 min at maximum speed to pellet the bacteria (16,000 x g). In order to do this, set your P-1000 to 750 l and deliver this volume of the saturated culture into the eppendorf tube twice. Make sure to label your eppendorf tubes with your name. After centrifugation, discard the supernatant by aspiration using a vacuum pump equipped with a Pasteur pipet and sterile pipet tip. DO NOT suck up the pellet!! It is safer to leave a little LB broth on top of the pellet, as opposed to sucking some of it up during this procedure. After aspiration, add another 1.5 ml of saturated liquid culture into the same tube and recentrifuge again at 16,000 x g. This is known as ‘double-pelleting’ and it will increase your overall plasmid DNA yield at the end of the procedure. Aspirate again and continue to the next step

what does step 1 do (lab 2)

This part of the procedure collects the bacterial cells which are suspended in the liquid medium into a cell pellet at bottom of the tube

step 2 of Plasmid DNA isolation (lab 2)

Add 250 µl of Buffer P1 (50 mM Tris-HCl at a pH 8.0, 10 mM EDTA, 100 µg/ml RNase A) to the tube and re-suspend the cells by vortexing or pipetting. It’s very important that the cell suspension in homogenous and no clumps are visible

what’s in buffer p1 (lab 2)

50 mM Tris-HCl at a pH 8.0, 10 mM EDTA, 100 µg/ml RNase A

what does step 2 do in lab 2

The cells are now re-suspended in a buffered solution with RNase. When the cells are lysed in the next step, the RNase will catalyze hydrolysis of all the RNA molecules into nucleotides, but the DNA will not be affected

step 3 in lab 2

Add 250 µl of Buffer P2 (1% SDS, 0.2 M NaOH). Close the cap and mix the solution rapidly by inverting 4-6 times.

what’s in buffer p2 in lab 2

1% SDS, 0.2 M NaOH

what does step 3 in lab 2 do and what do the reagents in buffer p2 do

SDS represents an acronym for the molecule sodium dodecyl sulfate. It is an ionic detergent which disrupts cell membranes and destabilizes all hydrophobic interactions holding various macromolecules in their native conformation. The high pH of the 0.2 M NaOH also denatures the macromolecules by changing the condition of ionizable groups (ionizing certain groups and deionizing others). The clearing you see is because the cells are lysing. The viscosity of the solution is increased by the increase in concentration of the macromolecules in the solution (a result of cell lysis)