Restriction Digest

Question 1

Purpose of Restriction Digest:

Answer 1

To confirm N1 plasmid

Question 2

What are restriction enzymes?

Answer 2

• found in bacteria as a natural defense to foreign, non-self DNA
• they cut “non-self DNA”
• they produce sticky ends (single strand overhang) or blunt ends

Question 3

How do restriction enzymes work?

Answer 3

• enzymes recognize and bind to specific sequences of DNA (restriction sites)
• once target site is found → restriction enzyme will make a double stranded cut in the DNA

Question 4

Sticky Ends

Answer 4

single stranded DNA “overhands” → usually preferred

EX: EcoRI cuts like this

Question 5

Blunt Ends

Answer 5

harder to work with

created when restriction enzyme cutes straight down the middle of a target sequence and leaves no overhang

Ex. SmaI does this

Question 6

Applications of Restriction Digest

Answer 6

Confirmation of plasmid

making recombinant proteins

DNA mapping

Gene Editing

Question 7

Work Flow of Restriction Digest:

Answer 7

• isolate plasmid from bacteria
• digest plasmids with restriction enzymes
• resolve digest by agarose gel and determine banding patterns

Question 8

Things to think about when choosing restriction enzymes to use:

Answer 8

• Determine if there is a restriction site within your insert
  
  • if using the recombinant plasmid in later applications make sure the restriction sites flank the insert and do not cut within your insert
• If you need to insert your gene in a specific region of the plasmid, make sure your restriction enzyme does not cut elsewhere in the plasmid

Question 9

Predicting Band Patterns

Answer 9

• Verify total plasmid size-or-insert and backbone size

Question 10

What happens if you only cut the plasmid once?

Answer 10

it linearizes

Question 11

What happens if you cut the plasmid twice?

Answer 11

you take out a chuck (makes 2 pieces)

Question 12

Problems with Restriction Digest

Answer 12

• Star Activity → random cutting
• Methylation

Question 13

Problems with Restriction Digest: Star Activity

Answer 13

when restriction enzymes are capable of cleaving sequences which are similar but. not identical to their defined recognition sequence, cleaving nonconical sites, usually occurs under certaine extreme conditions

Question 14

Preventing Star Activity

Answer 14

limit concentration of enzyme

optimize buffer (ionic strength/pH)

shorten reaction time

Question 15

Problems with Restriction Digest: Methylation

Answer 15

Some restriction enzymes are sensitive to DNA methylation states, cleavage can be blocked/impaired if recognition sites are modified

Endogenous DNA will be methylated however DNA fragments from PCR will not be methylated

Question 16

Examples of Methylation

Answer 16

sensitive enzymes → Dpn1 and Hpall

Question 17

How to choose an enzyme for restriction Digest:

Answer 17

Pick one that cuts in the insert (gene)

pick one that cuts outside the gene in the vector

Question 18

Why use Sal1 and Not 1 for Notch1-GFP Plasmid Restriction Digest?

Answer 18

Sal 1 → cuts twice

Not1 → cuts once

both have recognition sites within the Notch1 gene → can be used to check if the plasmid contains the Notch 1 gene

Sal 1 also cuts in the vector

Question 19

How do you know if the Restriction Digest confirmed the plasmid you are looking for?

Answer 19

if you see the bands you calculated it is correct

plasmid is correct → if Notch1 gene is within the plasmid

plasmid is incorrect → Notch1 gene is absent