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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

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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
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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

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4
Q

4 key properties of P2X2 receptors

A

require ATP to open

modulated by membrane potential, pH, and Zn

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5
Q

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

A

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

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6
Q

histidine residues of P2X2

A

involved in proton and zinc binding

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7
Q

cysteine residues of P2X2

A

disulfide bonds and bind zinc

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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)

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9
Q

site directed mutagenesis

A

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

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10
Q

which amino acids are often used for mutagenesis

A

alanine and cysteine

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11
Q

alanine substitutions

A

indicates amino acid importance

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12
Q

cysteine substitutions

A

used to determine amino acid topology

can see whether the cysteine is located intracellular or extracellularly

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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

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14
Q

what are the components of PCR reaction

A

dNTPs, buffer, template DNA, primers, Pfu turbo

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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

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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
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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)

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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
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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

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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)

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21
Q

resting membrane potential for neuron

A

-70 mV

inside neuron is more negative than outside

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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
what did primer design do
create mutation site added/subtracted a restriction enzyme site
26
what is special about XL1B cells
ligate the DNA to form circular DNA from mutant linear
27
before minipreps, what do colonies contain
both bacteria chromosomal DNA and plasmid DNA minipreps get rid of bacteria DNA and isolate plasmid DNA
28
miniprep steps (Qiagen kit)
1. resuspend 2. lyse 3. neutralize 4. clean DNA 5. elute DNA
29
buffer P1 is used for what
resuspension buffer
30
P1 buffer contains what
**Tris**: buffer that mantains pH **EDTA**: blocks DNAse activity, protects DNA **Glucose**: maintains osmotic pressure **RNAse**: degrades RNA
31
buffer P2 does what
lyse bacteria
32
buffer P2 contains what
**SDS** (detergent): breaks open cell membrane and denatures proteins **NaOH**: denatures genomic and plasmid DNA from double to single strand
33
what does N3 do
neutralize solution plasmid DNA reanneals but genomic DNA does not
34
buffer N3 contains what
**acid**: neutralizes base **high salt concentration**: causes denatured proteins, cellular debris, chromosomal DNA, and SDS to precipitate
35
why is spin column
DNA binds to silica column, PE wash buffer contains EtOH to wash away salts, elution buffer elutes
36
restriction enzymes
each recognizes a specific sequence of nucleotides and cuts most recognition sequences are palindromes (like GAATTC)
37
restriction enzyme: types of cutting
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
isoschizomers
restriction enzymes that recognize same sequence but cut it in different ways
39
what joins compatible cut ends of DNA
DNA ligase
40
PhoI
causes blunt ends has an isoschizomer digests at 75oC use cutsmart buffer
41
choosing restriction enzymes
* restriction site should flank insert, but not cut w/in insert * same restriction enzymes can cut your recipient plasmid
42
what do we use to determine concentration of DNA
spectrophotometric
43
miniprep low yield troubleshooting
* 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
miniprep low ratio troubleshooting
* 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
transformation efficiency equation
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
3 components of e-phys set up
digitizer, amplifier, computer
47
steps of ephys
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
2 wires in ephys set up
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
input signal generated w/in cell means we need to do what w/ amplifier/digitizer
need **analog in** to connect amplifier to digizier
50
if resting potential says -3.0, what needs to be changed
scale factor in lab bench (signal needs to be amplified more)
51
if amplifier says -21 but computer says 0mV, what is wrong
connection b/w digitizer and amplifier problem signal not being digitized (not amplifier issue)
52
liquid junction potential is recorded when... what are the junctions
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
liquid junction potential: why is Cl silver wire used
used to prevent interaction w/ 3M KCl (prevents Cl from KCl sticking to electrode)

MCDB 423 Neurobio Lab (1 decks)

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