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Microbio Lab 9
Colony PCR and HRP Overlay Assay
| Question | Answer | |
|---|---|---|
| what does PCR NOT tell you? | whether there is a mutation in the HRP coding sequence | |
| if there is no PCR product | the strain is a dud. it has the zeocin resistance gene but it does not have the HRP gene | |
| ___ + ___ = blue product | HRP + TMB substrate | |
| what does blue color indicate? | HRP expression. | |
| what regulates the transcription of the MAT-alpha HRP | AOX1 promoter | |
| what induces the AOX1 promoter? | methanol in the medium only and repressed by presence of glucose | |
| T/F: strains that turn blue on YNM should have a PCR product | true | |
| what does a darker blue color indicate? | more HRP coding sequence | |
| what do we use gel electrophoresis for? | to determine if our selected zeocin-resistant colony was a stud or a dud (presence of gene) | |
| what is a stud | a colony that contains the HRP gene | |
| what is the purpose of the HRP overlay assay? | to determine which colonies expressed HRP (functionality of gene) | |
| what is a YND plate?* | yeast nitrogen dextrose. should the colony grow? | no |
| YNM plate?* | yeast nitrogen methanol plate. should the colony grow? | yes if it has the HRP gene. it it does not, the colony is a dud. |
| what size should the PCR product be? | about 1000bp | |
| what if nothing shows up on PCR plate? | then you have a dud colony: no HRP gene | |
| what does a white colony mean? | no gene or gene not expressed | |
| what are the ingredients to making root beer? | water, brown and white sugar, champagne yeast, and root beer extract | |
| How much space and why should you leave?* | 1.25 inches: if too little space ... if too much... | soda will be flat. soda will overcarbonate. |
| what are possible contaminants? | Lactobacillus and Lactococci | |
| what happens if you leave your bottle out at room temp for too long? | you will get more ethanol and a lot more CO2 so explosion! | |
| what makes soda bubbly? | carbon dioxide | |
| what are the two formulas? | pyruvate -> acetaldehyde + Co2 Acetaldehyde + NAD+ --> ethanol + NADP | |
| why is the bottle hard? | pressure from CO2 | |
| what happens when the bottle is put in the fridge? | 1) fermentation stops 2) yeast settles out 3) flavor matures | |
| what is a PCR product* | region of HRP gene amplified by the 2 primers in PCR. if your sample contains this... | your cells have the HRP gene |
Created by:
syeonie
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