Rec' DNA Lab Week 2

1
Rec. DNA Lab Week 2

• Restriction Digestion and Agarose Gel
  Electrophoresis

2
Overview

• Assay our Plasmid preps from last week by
  digesting a portion of the prep with restriction
  endonucleases.
• These digests will then be run on an agarose
  electrophoresis gel and stained with Fast Blast
• This Lab (Ex. 4) and Thursdays lab (Ex. 6) will
  be combined into one write-up

3
Restriction Endonucleases

• Enzymes that recognize specific sequences of DNA
  and cleave the sugar backbone
• Originally named for the restriction of phage
  growth in certain bacterial strains
• Methylases/nucleases
• 1968 H.O. Smith, K.W. Wilcox, and T.J. Kelley
  isolated and characterized the first sequence
  specific restriction nuclease

4
Restriction Endonucleases

• Type II - Cuts at or near a short recognition
  sequence. Separate enzyme methylates the same
  recognition sequence.
• Palindromic vs. non-palindromic rec.sites
• Blunt vs. Sticky ends
• Type I - Cuts nonspecifically and far from
  recognition sequence. Contains both restriction
  and methylation activities.
• Type III - Cuts 24-26 bp downstream from a short,
  asymmetrical recognition sequence. Requires ATP
  and contains both restriction and methylation
  activities

5
Using Restriction Endonucleases

• Keep cold!
• Enzymes loose activity at room temperature
• Pipette from the surface of the liquid
• Dipping into the enzyme gets too much on the tip
• Different restriction endonucleases use different
  buffers- make sure you use the right one
• Buffers are usually 10X, add 1 ul for each 9 ul
  of total reaction volume

6
Agarose Gel Electrophoresis

• A gel matrix that uses an electrical current to
  separate molecules based on mass/charge rations
• Because DNA fragments have the same charge, they
  are separated by mass
• Larger fragments move slower, smaller move faster

7
Using our gel rigs

• Cool agarose for several minutes before pouring
• Caulk the edges of casting tray before filling
• Be careful removing and moving gels, they are
  slippery and fragile

8
Fast Blast Staining Procedure

• Wearing gloves, remove the gels from their trays
  and submerge them in the 100x stain in an
  appropriately sized container. Stain the gels for
  23 min, but not more than 3 min.
• Save the used 100x stain for future use. The
  stain can be reused at least 7 times.
• Transfer the gels to a large container containing
  500700 ml of clean warm (4055C) tap water.
  Gently shake the gels in the water for 10 sec to
  rinse.
• Transfer the gels to a large container with
  500700 ml of clean warm tap water for 5 min
• Move the gels gently in the water once every
  minute.
• Perform a second wash in clean warm water as in
  previous step

9
Changes to the lab

• pBR322 will not be used, substitute your purified
  plasmid instead - pGEM
• Use 150 mL of 0.8 agarose instead of 80 mL as
  the book says
• Use the buffer that is specific for each
  restriction endonuclease
• Endonuclease NdeI will replace PstI
• Fast Blast stain after electrophoresis instead of
  Et-Br

10
Checklist

• Per Table
• Set up tubes A-F and digest with restriction
  enzymes
• Remember to save tube BYou will need this in
  future labs!
• Run gel of digests and photograph
• Photographs will be available for download from
  Web CT after class
• Combine Ex 4 and 6 in one lab report
• Include answers to questions 1-8 in Ex. 4
• Thursdays lab
• review gels and prepare media for use in future
  labs
• Environmental Isolation Round II
• Introduce genomic DNA purification protocol
  handout
• Turn in Exercise I Write-up