Lab Techniques + Experiments W1-6

Question 1

What is the purpose of PCR?

Answer 1

To amplify the amount of DNA fragments

Question 2

First step of PCR + why?

Answer 2

Heat to 95 deg C to break the H-bonds between nitrogenous bases

Question 3

Second step of PCR + why?

Answer 3

Cool to 50-65 deg C to allow primers to anneal

Question 4

Why is there 40-60% CG bases in a primer and not more?

Answer 4

Would increase melting temperature for when they need to be removed

Question 5

Why do primers avoid repeating sequences and being complementary to each other?

Answer 5

If complementary, they stick to each other. If repeating, likely to stick to themselves

Question 6

Third step of PCR + why?

Answer 6

Heat to 72 deg C to initiate Taq polymerase extending on the primer

Question 7

What is special about Taq polymerases’ working conditions?

Answer 7

Can work at very high temps as is thermostable

Question 8

What is the purpose of electrophoresis?

Answer 8

Separating fragments of DNA of varying lengths to identify them

Question 9

How to make the 1% agarose gel for electrophoresis?

Answer 9

1g agarose + 100 ml of TAE buffer

Question 10

What is typically used as a counter stain in electrophoresis + why?

Answer 10

Ethidium bromide, intercalates between bases to show banding

Question 11

Why is the sample of DNA fragments mixed with glycerol?

Answer 11

Makes it sink

Question 12

What is a DNA size ladder used for?

Answer 12

Comparison of sample to ‘normal’

Question 13

Which pole do the fragments move towards and why?

Answer 13

+ve because phosphate backbone of DNA strand is -ve

Question 14

What does it mean that size is inversely proportional to distance?

Answer 14

The bigger the fragment, the less distance that fragment moves

Question 15

What is the outcome of making recombinant DNA?

Answer 15

Produces a plasmid or something similar that can be taken up into the cell and integrated which contains the target gene

Question 16

What do restriction enzymes do?

Answer 16

Cut plasmid and fragment at specific site to leave complementary sticky ends

Question 17

What does ligase do in forming recombinant DNA?

Answer 17

Joins DNA fragment and plasmid sticky ends

Question 18

What can be done once the target gene is in the plasmid, and what needs to be done to ensure each cell incorporates the gene?

Answer 18

Transformation into E.coli bacteria, allow for many cell replications and then cells will contain target gene

Question 19

Step 1 of Q-RT PCR

Answer 19

Extract all mRNA from cell/tissue + reverse transcribe to cDNA

Question 20

Step 2 of Q-RT PCR

Answer 20

Add PCR mix (DNA pols, buffer, fragments, primers) and run PCR

Question 21

What is the purpose of Q-RT PCR?

Answer 21

Detects and quantifies mRNA present in cell/tissue, shows gene expression

Question 22

How is the PCR product measured in Q RT PCR?

Answer 22

Brightness of intercalating dye

Question 23

What does it mean if there is a high conc of cDNA

Answer 23

High gene expression

Question 24

What is the purpose of CRISPR?

Answer 24

It is a form of gene/DNA editing that specifically targets certain sequences

Question 25

1st step of CRISPR

Answer 25

Viral vector carrying spacer sequences binds to cell + injects contents

Question 26

2nd step of CRISPR

Answer 26

Spacer sequence integrates into the genome between repeat sequences

Question 27

3rd step of CRISPR

Answer 27

Spacer transcribed to CRISPR RNA (guide RNA) which binds to a Cas protein

Question 28

4th step of CRISPR

Answer 28

Cas9 and gRNA targets homologous DNA and Cas9 causes dsDNA cuts in viral genome

Question 29

What does cutting the dsDNA cause?

Answer 29

Known mutations, inactivation of a gene

Question 30

What is used as a template in HCR after CRISPR cuts DNA?

Answer 30

Synthetic plasmid

Question 31

Lineage tracing with CRISPR (zebrafish example) step 1

Answer 31

Take gRNA for GFP gene and inject into embryo so it now has Cas19 and gRNA

Question 32

Lineage tracing with CRISPR (zebrafish example) step 2

Answer 32

dsDNA break occurs when gRNA finds GFP gene and this is fixed by NHEJ which creates mutations

Question 33

Lineage tracing with CRISPR (zebrafish example) step 3

Answer 33

Lets embryo develop and mutations are inherited

Question 34

Lineage tracing with CRISPR (zebrafish example) step 4

Answer 34

Compare scar barcodes, if share many scars then closely related and vice versa