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I. An overview of the Bio Lab 

 pipet protocol

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  • Pipette Etiquette
  • USE A LOT OF TIPS. WHEN IN DOUBT CHANGE THE TIP.
  • do not leave the lid of pipet tip holder open after grabbing new tip.
  • units we used in the lab: mainly micro-liters
  • Microliter – µ1 -.000001
  • Liter – L – 1

 

 

 

 

 

 hot plates 

  • rapid heating, heat shocking – (thermometer must be in water)

 incubator

  • an enclosed apparatus providing a controlled environment for the growing of microorganisms under controlled conditions

 autoclave 

  • performs sterilization through steaming/heat

*IMPORTANT: keep thing sterile in the lab, gloves – don’t touch face, mouth etc./pipet etiquette/do not drop things on the floor 

centrifuge

  • machine with a rapidly rotating container that applies centrifugal force to its contents, to combine or settle 

 


II. Overview of our first project in the lab

In this lab, we take DNA sequences for GFP (Green Fluorescent Protein) from fluorescent jellyfish, and catalyst its production; then combine with E Coli. cells – in theory, producing colonies that would soon fluoresce under black light.

 

Alba the rabbit 

 

 


 

III. Protocol

 

1. Digestion

Refer to the Digestion+Ligation+Transformation Protocol worksheet 

*snowflake symbol means keep on ice

A. GFP PLASMID

  1. Begin with  DNA – the GFP (combined with the RBS and Terminator) on a plasmid with chloramphenicol resistance 
  2. Add CutSmart buffer to help the reaction
  3. Add Enzymes  (PstI & XbaI)

The restriction enzymes cut the DNA at the site of a specific sequence.
Repeat these steps in separate tube for each color of fluorescent protein (GFP, YFP, RFP)

B. LACI PLASMID

  1. Begin with DNA –  LacI Promoter on a plasmid with Ampicillin resistance
  2. Add CutSmart buffer to help the reaction
  3. Add + Enzymes (PstI & Spel)

Incubate tubes at room temperature for 1 hour

Heat using hotplate for 20 min @ 65°C (kills enzymes)

2. Ligation

Ligation is done on ice.

Repeat these steps in separate tube for each color of fluorescent protein (GFP, YFP, RFP)

Combine-

  1. digested DNA ( RBS + GFP + terminator)
  2. digest DNA – Lacl promotor (+ampicilin-resist)
  3. Ligase buffer (contains ingredients essential to ligase activity)
  4. ligase (enzyme used catalyze the joining of two molecules by forming a new chemical bond)

incubate @ 16°C overnight – or- @ room temp. 10min

heat inactivate, with hotplate, @ 65°C for 10min

chill on ice in prep for transformation (or in -20° freezer for storage)

 

 

 

 

3. Transformation

*Keep on ice

Prepare tube with – 

  1.  (dh5α competent) E. coli cells
  2.  assembled GFP plasmid 

Incubate on ice for 20min.
Heat shock @ 42°C for 45sec
Chill on ice 2+min

Combine –

  1. +transformed E. Coli cells
  2. +SOC Media (maximizes the transformation efficiency of competent cells)

 Incubate @ 37°C for 2 hours in shaker.
 Pipette 200μL onto petri plates with LB agar + Ampicillin
 Incubate overnight (14-18hr) @ 37°C
 Check for growth and protein production (colonies appear as dots)
 Transfer plates to 20°C refrigerator.

 

4. CULTURE SINGLE COLONY in liquid media 

in a glass test tube, combine

  1. LB liquid media
  2.  transformed E.Coli colony

Incubate overnight in shaker at 37 C


IV. Additional Resources

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~emma w

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