Microbial detectives Flashcards
What are the risks associated with this practical?
- Tubes, pipette tips or wooden sticks that have been in contact with the live organisms should be placed immediately after use into disinfectant containers or in the autoclavable bins.
- Bunsen burner flames are a potential hazard. Don’t leave unattended
- Acetone is flammable. Keep away from the Bunsen flame. When performing gram stains and spore stains be aware of others around you in the laboratory to avoid accidents
what names do we normally refer to bacteria by?
by their binomial genus and species names
What is the definitive method of identifying bacteria and fungi?
to determine the complete genomic DNA sequence of each isolate
What are the components we need for PCR?
- DNA template containing original sequence of DNA to be amplified
- Two DNA primers (forward and reverse) which target specifically the fragment to amplify
- Taq DNA polymerase
- dNTPs (deoxynucleotide triphosphates of adenine, guanine, thymine and cytosine
- Reaction buffers (often including MgCl2)
- Water (to ensure final concentrations are accurate)
- Loading buffer for the gel (not usually added to PCR mix but kit we are using includes it)
What are the three major steps in PCR?
- Denaturation at 90-95 degrees for 15s-1 min. Double stranded DNA melts open into single-stranded DNA molecules
- Annealing around 50-60 degrees for 45 secs (temp dependant on primer sequences). Drop in temperature allows the primers to find their complementary sequences on the single-stranded template and bind (anneal) through base complementarity. The templates strands also re-anneal but the primers are added in excess to out-compete other annealing events
- Extension at 72 degrees for 1-2 mins: the primers are in place so can ‘prime’ the synthesis of a new strand by providing a short sequence of double stranded DNA for the Taq polymerase to extend from and build a new complementary strand
How many times are the 3 steps in PCR repeated?
30 cycles or more
what is PCR achieved using?
achieved by automated heating and cooling of the tubes containing the reaction mixture in a programmable thermal cycler
What is the DNA fragment to be amplified determined by?
Where the primers bind
What are primers?
short (18-30 bp long) synthetic DNA strands that are complementary to the beginning and end of the DNA fragments to be amplified. Both primers must bind the genome only once
What is the ideal working temperature to Taq polymerase?
72 degrees
What does Taq polymerase do?
adds on the complementary dNTPs one by one to the template in the 5’-3’ direction. The Taq will stop adding dNTPs when there is no template left to read or when the extension time is over. The extension time is determined by the length of the fragment to be amplified.
What do universal yeast primers do?
bind specifically to the internal transcribed spacer regions found in eukaryotic ribosomal DNA (rRNA). These primers only bind to eukaryotic rRNA so they can be used to distinguish yeast isolates from bacterial
Why can PCR of the whole region (between yeast primers ITS1 and ITS4) be used to distinguish between different yeasts?
Because in yeast the size of the spacer ITS vary between species and genera
give features of PCR tubes?
- tiny and hold a maximum of 0.2ml
- have thinner walls than other microcentrifuge tubes to allow efficient heat transfer
What is put in the PCR tube for the yeast PCR?
- Two yeast DNA primers (ITS1 and ITS4)
- Taq DNA polymerase
- dNTPs
- reaction buffer
- MgCl2
- Gel loading buffer
- Water
What are the six different tubes set up in the yeast PCR?
- Negative control (add 2ul water but no DNA or isolate)
- Positive control (add 2ul yeast DNA -provided on ice)
- Isolate A
- Isolate B
- Isolate C
- Isolate D
How should you obtain the colony for the PCR mix?
When setting up A-D wooden spatulas should be used to scrape a small amount of colony from the growth plate and add directly to the PCR reaction mix in the thin-walled tube. Only very little is needed otherwise the PCR is inhibited by the cell-wall components of the sample.
How would water and purified yeast DNA be added to the PCR mix?
using a pipettor and a disposable plastic tip
What would you expect to see after electrophoresis of your PCR samples?
- Expect to see nothing in negative control lane
- Expect to see bases in positive control lane
- Expect to see nothing in bacterial isolates
- Expect to see the same as the positive control lane in yeast
How do you carry out the agarose gel electrophoresis for the yeast PCR?
• When the PCR has finished 10 ul of each PCR component is loaded on a 2% agarose gel
- The gel is placed into the electrophoresis tank and submerged so that the level of electrophoresis buffer is about 2m above the gel. The gel contains SafeView which is a skin irritant
- DNA markers (‘M’) are pipetted in Lane 1. These are molecular weight markers that can be used to calibrate the gel and serve as standards to estimate the size of the unknown DNA fragments
- The PCR mix including loading buffer: this contains glycerol to ensure that the DNA samples sink into the wells in the agarose gel, and a red dye, which acts as a marker dye to indicate the progress of electrophoresis. PCR sample (10ul) is loaded into separate lanes of the gel.
- When all the samples are loaded the samples are electrophoresed at 80-100 V until the dye has migrated to just before the end of the gel. This takes 30-45 min. After this the gel is taken out of the buffer and placed on a UV transilluminator to visualise the DNA and is photographed (gloves must be worn when handling the gel)
What do you know if the electrophoresis markers are resolved?
Electrophoresis has worked
Why may your positive control not show?
may because the enzymes are too old or you forgot to put primers in
How does a bacterial or fungal cell divide on an agar plate?
exponentially
