Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
7 Cards in this Set
- Front
- Back
|
what is the process of purification of genomic DNA
|
1. transfer the sample to a tube
2. grind the sample to break tissue 3. add lysis buffer (proteinase k, RNAse, EDTA) 4. incubate at 55 degrees celcius for 16 hours 5. transfer lysate into DNA spin column 6. centrifuge for 3 mins at full speed. 7. pour out the flow through 8. wash spin column 4 times with wash buffer 9. spin the empty column to remove any wash buffer left 10. put the spin column in a centrifuge tube 11. add nucleas free water to the tube and incubate for 2 mins 12. spin the column 13. KEEP THE FLOW THRU 14. add another 250ml d.i water to the column 15. spin again 16. keep the flow thru |
|
|
what does proteinase k do?
|
degrades protein
|
|
|
what does RNAse do?
|
degrades RNA
|
|
|
what does EDTA do?
|
removes cations and inactivates DNases
|
|
|
why do we centrifuge for 3 mins at full speed
|
to separate the plasmid DNA from the flow through. the plasmid binds to the column and the flow through goes into the bottom
|
|
|
what is the point of washing the flow through with wash buffer 4 times?
|
these extra washes should help further remove any protein, RNA, and lipids that are still in the DNA
|
|
|
why must any residual wash solution be removed from the spin column?
|
any residual wash solution left in the column has ethynol in it which could interfere with further DNA manipulation.
|
