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Flashcards in Biotech lab 5 Deck (19)
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1

After adding ammonium bicarbonate (50mM) to 10 mL of ddH2O, what pH should be observed?

8-9

2

What is in the destaining solution?

1 mL of acetonitrile and 1 mL of 50 mM ammonium bicarbonate

3

What band is taken from the SDS-PAGE gel?

the PTEN band, minimize gel amount and use clean scalpel

4

What is added to the gel band?

destain solution

5

Once the destain solution is added to the gel band, what is done?

bench top incubator at 37C for 15 minutes until band becomes clear

6

How is the gel dehydrated?

adding 300uL of acetonitrile

7

what does acetonitrile do?

reduce hydrophobic interactions of CBB and gel

8

after wash with acetonitrile, where is the gel piece put?

speed vacuum for 20 minutes

9

what protease is used?

trypsin

10

what is in the trypsin digestion buffer?

resuspended trypsin, ammonium bicarbonate

11

how is the sample rehydrated?

adding enout trypsin digestion buffer to cover the gel piece

12

the gel and digestion buffer is incubated where?

on ice for 10 mins

13

how will you know that the trypsin has entered the matrix?

gel piece should swell

14

why is parafilm added after the on ice incubation?

to minimize evaporation

15

where is the gel sample incubated after adding parafilm?

bench top shaking incubator for 2 hrs at 37C

16

if gel isnt covered, what do you add?

ammonium bicarbonate

17

How often do you check the gel?

every 30 minutes

18

after the incubation, what is added for peptide extraction?

vortex and spin down, add formic acid solution and collect supernatant

19

After collecting supernatant what do you do?

add 1 ul of sample in maldi pate and add 1 ul of UV absorbing maldi matrix

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