Download BIOT 3 Lab 3 Handout 1

Name_____________________________
1/5/2016 – Biotech 3
Lab Day 3:
Part I – Plasmid Miniprep
In the previous lab we isolated plasmid DNA from a bacterial culture. Unfortunately, after reviewing
everyone’s DNA concentration values, the amount of plasmid DNA that was recovered was lower than
desired. Typically, most minipreps would yield a plasmid DNA concentration of 100 – 200 ng, even up
to 500 ng/μl. Everyone in the class obtained a plasmid DNA concentration somewhere near 20 ng/μl.
The good news is that everyone performed a successful miniprep by purifying and obtaining plasmid
DNA at the end of the experiment. Also, everyone obtained an A260/A280 ratio above 1.8, indicating that
the miniprep was not contaminated with protein. However, the lower than desired final concentration of
plasmid DNA may hinder the quality of downstream experiments. For example, visualization of DNA
bands after running gel electrophoresis may be difficult due to the low DNA concentration. Therefore,
before continuing with restriction enzyme digests on the pET3a plasmid, we’ll repeat the miniprep and try
to obtain a higher plasmid DNA concentration. We’ll need to trouble shoot our experiment and try to
obtain a higher yield of plasmid DNA. One improvement that we can make is to use a larger volume of
bacterial culture to pellet for our miniprep extraction, i.e. the more bacteria, the more plasmid. Second,
we will use a fresh overnight culture rather than an older culture that had been stored in the fridge (4 °C)
for over 24 hours. Older bacterial cultures may lose their selective plasmid if the antibiotic is no longer
present, for example if the antibiotic in the culture broth has been degraded over time. A third remedy will
be to use an alternative miniprep kit from a different manufacturer. Miniprep spin columns are made by
various manufacturers and thus the resin that traps plasmid DNA during purification can vary in binding
capacity parameters as well as shelf life. In the previous lab section we used a miniprep kit from
Clontech Laboratories Inc.
In this lab you’ll re-isolate the plasmid pET3a from a larger sample of a freshly grown overnight E. coli
culture using a miniprep kit from Qiagen Inc. The instructions are very similar to what you have already
done.
Instructions:
1. Obtain 8 ml of a saturated E. coli XL-1 pET3a LB culture. Pipette 1.5 ml of the
cell culture into two 1 ml microfuge tubes.
2. Spin the tubes in a standard benchtop centrifuge for 1 minute at 11,000 x g (full
speed).
3. Discard the supernatant by decanting into a liquid waste container, remove as much
liquid as possible without disturbing the pellet.
4.
Repeat centrifugation on the remainder of the E. coli culture using the same
microfuge tubes. Observe that the pellet is increasing in size after each
centrifugation step.
5. Resuspend cells: Resuspend pelleted bacterial cells in 250 μl of Buffer P1 and
transfer to a microcentrifuge tube.
Name_____________________________
1/5/2016 – Biotech 3
6. Lyse cells: Add 250 μl Buffer P2 and mix thoroughly by gently inverting the tube
4 – 6 times. Incubate the tube at room temperature for 5 minutes (do not incubate
for longer than 5 minutes!).
7. Add 350 μl Buffer N3 and mix immediately and thoroughly by gently inverting the
tube 4 – 6 times (you may invert the tube more than 6 times in order to obtain
sufficient mixing).
8.
Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
9. Apply 800 μl of the supernatant from step 8 to the QIAprep spin column by
pipetting.
10. Centrifuge for 30 – 60 seconds. Discard the flow-through. Centrifuge again for 30
– 60 seconds.
11. We’ll skip the additional wash step. The additional wash step is not necessary for
E. coli XL-1 Blue strains. – we’ll do the wash step after all.
12. Place the QIAprep spin column in a clean 1.5 microfuge test tube. To elute DNA,
add 50 μl of Buffer EB to the center of the column, let stand for 1 minute, and
centrifuge for 1 minute.
Part II– Determine DNA Concentration
As soon as you have completed Part I and obtained your plasmid DNA, measure your sample’s plasmid
DNA concentration using the Nanodrop. Have one member take two measurements and then average the
values and report to the nearest significant figure as well as the A260:A280 ratio.
Calculate your plasmid DNA concentration using the formula below:
Concentration of DNA sample = A260 x 50 μg/ml x dilution factor
Part III – Restriction Enzyme Digest
Much of genetic engineering relies on the ability to move a genomic sequence from one DNA site to
another. Restriction enzymes (aka restriction endonucleases) were discovered in the late 1960’s by
Werner Arber, Hamilton Smith, and Dan Nathans. In nature, bacteria use restriction enzymes as a
defense mechanism to protect against infectious pathogens such as viruses called bacteriophage, also
known as phage. Phage viruses reproduce by injecting DNA into a host bacteria and then use the host’s
cellular machinery to replicate more copies of phage virus. Bacteria produce restriction enzymes that
cleave phage DNA, thus allowing the bacteria to survive a viral infection. Bacteria prevent their own
DNA from being digested through modifications of restriction site target sequences. One such protective
modification is called methylation where a methyl group is added to the DNA sequence, thus sterically
restricting a restriction enzyme.
Name_____________________________
1/5/2016 – Biotech 3
Restriction enzymes are typically named from their origin of isolation or discovery. For example, the
restriction enzyme EcoRI was isolated from the bacteria Escherichia coli strain RY13, HindIII was
discovered in Haemophilus influenza strain Rd, and PstI was discovered in Providencia stuartii.
Restriction enzymes separate or “cut” a DNA strand at a particular site, meaning each restriction enzyme
is specific for a particular DNA sequence, that sequence is called the restriction or recognition site.
Dependent on the enzyme, the cut in the DNA strand may either result in “blunt” ends with no unpaired
DNA base pairs, or the cut may result in “sticky” ends where there are overhangs of unpaired bases at
either the 5’ or 3’ ends. Typically restriction enzymes recognize palindromic sequences where the DNA
base pair sequence is the same forward as it is backwards on the complementary (opposite) DNA strand.
Restriction sites are often 4 – 6 bases long, though some restriction enzymes recognize sequences up to 12
bases long.
There are various manufacturers of restriction enzymes. One commonly used manufacturer is New
England Biolabs®, Inc. The amount of DNA that you cut depends on your application. Diagnostic
digests typically involve ~500 ng of DNA, while molecular cloning often requires 1-3μg of DNA. The
total reaction volume usually varies from 10-50μL depending on application and is largely determined by
the volume of DNA to be cut.
In this experiment, you will prepare 3 samples for your restriction enzyme digests and one reaction
control.
Restriction digest Instructions:
1. Calculate the quantity in μl of your plasmid that you’ll require to add 250 ng of plasmid into 30 μl
final volume. Enter the volumes for your restriction enzyme digests in the table below:
2. Have the instructor check your calculations for the quantity of plasmid DNA and water for each
reaction. Once you get the instructors approval, begin to make your sample reactions. Watch out
for pipetting errors, the volumes are small and therefore small errors may make a big impact!
3. Once you’ve completed each mixture, mix by gently pipetting up and down.
4. Spin the reaction tube in a microcentrifuge for 30 seconds at full speed.
5. Incubate the reaction tubes at 37 °C for 1 hour.
6. Spin the reaction tube in a microcentrifuge for 30 seconds at full speed.
Name_____________________________
7.
1/5/2016 – Biotech 3
Which enzyme buffer did we use for the digest and why was this particular buffer chosen?
8. What is the purpose of BSA?
Name_____________________________
1/5/2016 – Biotech 3
Part IV – Gel Electrophoresis
Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Every
molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and
analysis of DNA. We will use agarose gel electrophoresis to verify restriction enzyme digestion of the
pET3a vector.
Electrophoresis is a method of separating substances based on the rate of movement while under the
influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is
created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then
pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. During
electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and
negative electrode. The DNA to be analyzed is forced through the pores of the gel by the electrical
current. Under an electrical field, DNA will move to the positive electrode (red) and away from the
negative electrode (black). Several factors influence how fast the DNA moves, including; the strength of
the electrical field, the concentration of agarose in the gel and most importantly, the size of the DNA
molecules. Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself
is not visible within an agarose gel. The DNA will be visualized by the use of a dye that binds to DNA.
Instructions:
Preparing the agarose gel
1. Measure 1.25 g Agarose powder and add it to a 500 ml flask.
2. Add 125 ml TAE Buffer to the flask. (the total gel volume well vary depending on the size of
the casting tray)
3. Melt the agarose in a microwave or hot water bath until the solution becomes clear. (if using a
microwave, heat the solution for several short intervals - do not let the solution boil for long
periods as it may boil out of the flask).
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly.
5. Seal the ends of the casting tray with two layers of tape.
6. Place the combs in the gel casting tray.
7. Pour the melted agarose solution into the casting tray and let cool until it is solid (it should
appear milky white).
8. Carefully pull out the combs and remove the tape.
9. Place the gel in the electrophoresis chamber.
10. Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.
Loading the gel
1. Add 6 l of 6X Sample Loading Buffer to each 30 l restriction enzyme reaction
2. Record the order each sample will be loaded on the gel and ladder.
3. Carefully pipette 20 l of each sample mixture into separate wells in the gel.
4. Pipette 10 l of the DNA ladder standard into at least one well of each row on the gel.
Running the gel
1. Place the lid on the gel box, connecting the electrodes.
2. Connect the electrode wires to the power supply, making sure the positive (red) and negative
(black) are correctly connected. (Remember – “Run to Red”)
Name_____________________________
1/5/2016 – Biotech 3
3. Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on
the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes!
4. Check to make sure the current is running through the buffer by looking for bubbles forming on
each electrode.
5. Check to make sure that the current is running in the correct direction by observing the movement
of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the
DNA).
6. Let the power run until the blue dye approaches the end of the gel.
7. Turn off the power.
8. Disconnect the wires from the power supply.
9. Remove the lid of the electrophoresis chamber.
10. Using gloves, carefully remove the tray and gel.
Gel Staining (fyi we did not stain our gels using this method)
1. Using gloves, remove the gel from the casting tray and place into the staining dish.
2. Add warmed (50-55°) staining mix.
3. Allow gel to stain for at least 25-30 minutes (the entire gel will become dark blue).
4. Pour off the stain (the stain can be saved for future use).
5. View the gel against in a UV box.
6. Record the gel image.