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From 2010.igem.org

Meeting Minutes

[http://docs.google.com/present/edit?id=0Ae31et9w_tAhZGZyc3R2ejJfOGNnY2drMmZz&hl=en&authkey=CPHGzfIB Weekly Leader Presentation: Karina]

Agenda

Karina's Notebook

Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and (RFP + T) ligations on monday.

Part #'s:

• GFP: E0040 (cut at S and P)
  
• RFP: E1010 (cut at S and P)
  
• Term: B1006 (cut at X and P)
  
  

Recipe

• 12.0 uL DNA
  
• 1.0 uL each enzyme
  
• 5.0 uL NEB buffer 2
  
• 5 uL BSA (10x)
  
• Sterile H20 to fill up to 50 uL
  
  

Mix and put 37º waterbath for 2 or more hours.

Making Gel
To make 50 mL of 0.8% agarose gel, add:

• .4 g agarose
  
• 50 mL TAE (1x)
  
• 10 uL EtBr
  
  

Loading the Gel

• add 10 uL loading dye (6x) to digests
  
• add 40 uL of dye + digests to wells
  
• don't forget to include wells with controls! (uncut plasmids)
  
• load 10 uL ladder
  
• run at 95 V for about an hour
  

!!note sizes of plasmids/inserts
GFP plasmid (pSB1A2) --> 2079 bp
GFP insert --> 720 bp
RFP plasmid (pSB2K3) --> 4425 bp
RFP insert --> 681 bp
Terminator plasmid -->

Gel Results [add link to gel picture here]

All bands look fine except for, there were no bands for the terminators. They were so small they ran off the gell.