Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MCDB 423 Neurobio Lab > Midterm > Flashcards

Midterm Flashcards

1
Q

3 types of receptors in the nervous system

A

nicotinic receptor superfamily: pentameric

glutamate receptor family: tetrameric

ATP P2X receptor family: trimeric

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

steps of creating mutant receptors

A
  1. design primers
  2. PCR rxn
  3. Digest PCR product w/ DpN1, destroy methylated parental DNA
  4. Transform bacterial cells
  5. Plate onto LB/Amp (kill bacteria w/o plasmid)
  6. Pick colonies, miniprep
  7. Digest DNA to select for mutant
  8. Transcribe to make RNA
  9. Inject RNA into oocytes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

how many P2X ATP receptor genes in mammals

which are highly expressed in brain

A

7 genes

P2X2, P2X4, and P2X6 highly expressed in brain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

4 key properties of P2X2 receptors

A

require ATP to open

modulated by membrane potential, pH, and Zn

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

how do membrane potential, pH, and Zn affect P2X2 currens

A

more negative membrane potential, more zinc, more acidic pH = greater current

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

histidine residues of P2X2

A

involved in proton and zinc binding

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

cysteine residues of P2X2

A

disulfide bonds and bind zinc

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

open reading frame

A

sequence found b/w an ATG (Met) and a stop codon

there are 6 potential reading frames (which are translated into proteins)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

site directed mutagenesis

A

molecular bio method used to make specific changes to the DNA sequences of a gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

which amino acids are often used for mutagenesis

A

alanine and cysteine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

alanine substitutions

A

indicates amino acid importance

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

cysteine substitutions

A

used to determine amino acid topology

can see whether the cysteine is located intracellular or extracellularly

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

PCR steps

A

denaturation: temp increased to separate DNA strands

annealing: temp decreased for primers to base pair to complementary DNA template

extension: polymerase extends primer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what are the components of PCR reaction

A

dNTPs, buffer, template DNA, primers, Pfu turbo

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what are the three different types of PCR DNA segments

A

full length tempalte

segments that are in the process of extension (unknown length)

segments of fixed length equal to distance b/w primers

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

site directed mutagenesis: what makes a good primer

A
  • forward/reverse must be complementary
  • 25-30 bps in length
  • mutation should be in middle
  • GC content b/w 40-60%
  • look for secondary structures
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Pfu vs Taq polymerase

A

Taq: cheaper and faster; not good for sequences longer than 500-1000 bps, can add base to 3’ end causing frame shift mutation

Pfu: proofreading activity and higher fidelity (fewer errors)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

transformation of bacteria: steps

A
  1. amp resistant plasmids added to XL1 blue cells
  2. Cells heat shocked, cells take up plasmid and are transformed
  3. cells spread on agar plate containing ampicillin
  4. ampicillin kills cells w/o plasmid
  5. cells incubated
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

why are Xenopus oocytes ideal for ion channel expression

A

express large amounts of the protein

voltage clamp techniques are simple

oocytes are large and easy to see

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

limitation to oocyte expression system

A

large amount of calcium activated chloride channels, which could cover P2X2 channel responses (need to be silenced using inhibitor or reduce Ca in solution we use)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

resting membrane potential for neuron

A

-70 mV

inside neuron is more negative than outside

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

ion concentrations in cells and which whey they flow

A

Na: more concentrated extracellular

K: intracellular

Cl: extracellular

sodium potassium pumps 3 Na out, 2 K in

23
Q

methods of measuring potential of oocytes

A

insert electrode into cell and complete circuit w/ electrode in bath solution

24
Q

what bath solutions were used

A

ORS: measures resting potential

high potassium: caused depolarization

low chloride: caused hyperpolarization

25
Q

what did primer design do

A

create mutation site

added/subtracted a restriction enzyme site

26
Q

what is special about XL1B cells

A

ligate the DNA to form circular DNA from mutant linear

27
Q

before minipreps, what do colonies contain

A

both bacteria chromosomal DNA and plasmid DNA

minipreps get rid of bacteria DNA and isolate plasmid DNA

28
Q

miniprep steps (Qiagen kit)

A
  1. resuspend
  2. lyse
  3. neutralize
  4. clean DNA
  5. elute DNA
29
Q

buffer P1 is used for what

A

resuspension buffer

30
Q

P1 buffer contains what

A

Tris: buffer that mantains pH

EDTA: blocks DNAse activity, protects DNA

Glucose: maintains osmotic pressure

RNAse: degrades RNA

31
Q

buffer P2 does what

A

lyse bacteria

32
Q

buffer P2 contains what

A

SDS (detergent): breaks open cell membrane and denatures proteins

NaOH: denatures genomic and plasmid DNA from double to single strand

33
Q

what does N3 do

A

neutralize solution

plasmid DNA reanneals but genomic DNA does not

34
Q

buffer N3 contains what

A

acid: neutralizes base

high salt concentration: causes denatured proteins, cellular debris, chromosomal DNA, and SDS to precipitate

35
Q

why is spin column

A

DNA binds to silica column, PE wash buffer contains EtOH to wash away salts, elution buffer elutes

36
Q

restriction enzymes

A

each recognizes a specific sequence of nucleotides and cuts

most recognition sequences are palindromes (like GAATTC)

37
Q

restriction enzyme: types of cutting

A

can cut to make sticky ends or blunt ends

star activity: cleave noncanonical sites

methylation sensitive RE: cleave DNA at specific unmethylated cytosine residues

38
Q

isoschizomers

A

restriction enzymes that recognize same sequence but cut it in different ways

39
Q

what joins compatible cut ends of DNA

A

DNA ligase

40
Q

PhoI

A

causes blunt ends

has an isoschizomer

digests at 75oC

use cutsmart buffer

41
Q

choosing restriction enzymes

A
  • restriction site should flank insert, but not cut w/in insert
  • same restriction enzymes can cut your recipient plasmid
42
Q

what do we use to determine concentration of DNA

A

spectrophotometric

43
Q

miniprep low yield troubleshooting

A
  • choose colonies that are dense w/ bacteria
  • resuspend cells completely w/ buffer P1 before adding P2
  • if ethanol isn’t added to buffer PE, DNA will be washed out of filter w/ the flowthrough
  • elution buffer should be in pH range of 7-8.5
44
Q

miniprep low ratio troubleshooting

A
  • genomic DNA can contaminate final DNA product of lysis steps done incorrectly
  • faulty/old RNase in buffer P1 will prevent degradation of RNA and contaminate DNA
45
Q

transformation efficiency equation

A

TE = number colonies / ug plasmid DNA spread on plate

DNA spread on plate =

conc. of DNA (ug/uL) x volume of DNA (uL) x fraction of DNA spread on plate / total volume in tube

46
Q

3 components of e-phys set up

A

digitizer, amplifier, computer

47
Q

steps of ephys

A
  1. configure digitizer (make sure on 1500B not demo)
  2. configure telegraph instrument (computer is reading amplifier)
  3. define signals in “lab bench”
    1. units: mV or A
    2. scaling factor: how much amplifier is amplifying signal
  4. edit protocol
    1. sampling frequency: how often recording membrane potential
  5. Check input/outputs
48
Q

2 wires in ephys set up

A

1 is in bath solution, 1 is in cell

electrode in cell measures resting membrane potential, sends it to amplifier, then digitizer, then computer

49
Q

input signal generated w/in cell means we need to do what w/ amplifier/digitizer

A

need analog in to connect amplifier to digizier

50
Q

if resting potential says -3.0, what needs to be changed

A

scale factor in lab bench (signal needs to be amplified more)

51
Q

if amplifier says -21 but computer says 0mV, what is wrong

A

connection b/w digitizer and amplifier problem

signal not being digitized (not amplifier issue)

52
Q

liquid junction potential is recorded when…

what are the junctions

A

in low Cl and high K blanks (higher in low Cl)

need to minimize transfer of ions across junctions: bath and pipette, wire and 3M KCl

53
Q

liquid junction potential: why is Cl silver wire used

A

used to prevent interaction w/ 3M KCl (prevents Cl from KCl sticking to electrode)

MCDB 423 Neurobio Lab (1 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions