Extraction protokoll #64
Conversation
…nceCommunity.github.io into extraction_protokoll
_protocols/new-dna-extraction.md
Outdated
| - We assumed all pellets correspond to 80 mg and added 640µl Y-PER | ||
| - Mix by pipetting up and down until the mixture is homogenous | ||
| - Suspend cells: | ||
| 1. add an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. |
There was a problem hiding this comment.
Y-PER is a detergent optimized for yeast cell lysis. (https://www.thermofisher.com/order/catalog/product/78991#/78991)
| - Discard supernatant, | ||
| - Add 400μL of DNA Releasing Reagent A, | ||
|
|
||
| - Add 400μL of DNA Releasing Reagent A | ||
| - Add 400μL of DNA Releasing Reagent B | ||
| - Mix by pipetting up and down until the mixture is homogenous | ||
| - Incubate at 65°C for 10 minutes. (can be extended – SHOULD BE EXTENDED??) | ||
|
|
||
|
|
||
| ### STEP 4: Stop protein activity in the solution | ||
| - Add 200μL of Protein Removal Reagent to mixture |
There was a problem hiding this comment.
Protein Removal Reagent is probably either protease to digest proteins or a salt solution to precipitate protein (salting-out).
| - Add 600μL isopropyl alcohol to fill tube. | ||
| - Mix gently by inversion. (>20x). | ||
| - Separate genomic DNA by centrifuging the mixture at 13,000 × g for 10 minutes. | ||
| ### STEP 5: Separate the DNA from other molecules |
There was a problem hiding this comment.
DNA is negatively charged, therefore hydrophilic (dissolves in water). The carbon chain of alcohol is hydrophobic, so the DNA is less soluble and precipitates. Isopropanol has a longer carbon chain than ethanol (is more hydrophobic) and thus precipitates DNA stronger than ethanol.
The problem is that isopropanol also precipitates salts. To remove salts, we wash the DNA pellet with ethanol in STEP 6.
There was a problem hiding this comment.
We could add here a figure of Isopropanol and ethanol?
|
I add and commit only the new-dna-extraction.md. However, I have changes shown in the status in all kinds of documents. However, I did not do these changes. Should I do something about it? |
|
Will use the second extraction protocol PR to work a bit more: #85 |
Started but not finished the extraction protokoll!