Quiz 2 Info

Question 1

RT lab:

- prep: make … and make … (… + … + …)

Answer 1

reverse transcription reaction;

RNA sample; RNA; primer; water

Question 2

RT lab:

- prep: also have to … the RNA to … to … and get rid of any …

Answer 2

heat up the RNA;
70 degrees;
denature the RNA;
secondary structure

Question 3

RT lab:

- mix reverse transcriptase and RNA sample and let them … and then switch temp from 25 degrees to … degrees C

Answer 3

anneal;

42

Question 4

RT lab:

• mix reverse transcriptase and RNA sample and let them anneal and then switch temp from 25 degrees to 42 degrees C. 42 is where … and … step occurs
• at 70 degrees, … will be …

Answer 4

reverse transcriptase becomes active;
extension;
reverse transcriptase;
inactivated

Question 5

RT lab:
Gene specific primer only binds to …, only works well if sequence is … Otherwise, it may not be able to bind anything

Answer 5

that particular gene;

well expressed

Question 6

RT lab:

• oligo dT primer will bind to anything that has a … on it
• random primer: … primer with … sequence

Answer 6

poly A;
6 nucleotide;
random

Question 7

+RT is the experimental tube: contains … and …

Answer 7

RNA template;

reverse transcriptase

Question 8

For -RT, volume of reverse trans is replaced with … to make sure that we’re not copying from … or any other … in the sample

Answer 8

water;
genomic DNA;
DNA

Question 9

RT lab:

- for -RT, … in PCR. if there is a product, tells us that there’s … in the sample

Answer 9

shouldn’t see product;

genomic DNA

Question 10

RT lab:

-RT and -RNA are … controls bc we’re not expecting to see products in PCR for them

Answer 10

negative

Question 11

For +RT and -RT gel, expect to see … and … bands and … bands

Answer 11

28S; 18S; small RNA

Question 12

RT lab:
See RNA in pcr product for … and …

no rna should be present in PCR product for …

smear for +RT should be … than that of -RT –> should be … of nucleotide signal in -RT

Answer 12

+RT; -RT;
-RNA;
brighter;
no enhancement

Question 13

… RNA for PCR:

• product size will be about … bp
• should see nothing for -RT and -RNA on gel after PCR
• if genomic DNA in -RT, would see band at about … bp
• If -RNA was contaminated, could see bands …

Answer 13

linear ds;
1141;
2500;
anywhere along the gel

Question 14

RT lab:

we’re trying to amplify …, makes it important to know where … and … are
- if trying to amplify specific domain, this wouldn’t be important

Answer 14

whole coding region;

start; stop codons

Question 15

RT lab:

know which primer is upstream and which is downstream - check ppt

Answer 15

ok

Question 16

RT lab:

primers are …-… nucleotides in length bc they’re very … –> highly unlikely to just … find that sequence

Answer 16

18; 24;
specific;
randomly

Question 17

RT lab:
want to have primers with …-…% GC content
- keep GC and TA content …

Answer 17

40; 60;

relatively equal

Question 18

RT lab:

• higher annealing temperature: … primer binding, can’t …
• lower annealing temp - primers start …

Answer 18

less;
copy anything;
binding anywhere

Question 19

RT lab:

• GC clamp helps … and polymerase starts … from end
• having more than ..-… Gs or Cs can be problematic

Answer 19

3’ end bind to template;
copying;
2; 3

Question 20

RT lab:

• if primer forms … can’t form template
• if primer … with itself or with other primer, won’t be able to form template

Answer 20

hairpin;

hybridizes

Question 21

RT lab:
- avoid runs and repeats- having same nucleotide … or have same … –> by having repeated units, primer can … and won’t …

Answer 21

repeated;
set of two nucleotides;
slide on template;
start from the exact same place