Haynes Lab:Notebook/Engineering PC-TFs/2013/03/18

Summary

Cutting inserts to be placed in mammalian vector

• Procedure

A) Get biobricks and enzymes from freezer.

SpeI, PstI, XbaI

The following list of parts are:

• BL01
• BL05
• BL09
• BL12

  DNA          20.0 µL
  Enzyme 1     1.0  µL
  Enzyme 2     1.0  µL
  10x buffer   3.0  µL
  dH2O         5.0  uL
            
             = 30.0 uL  

Digest Order:

Biobrick Restriction Enzymes

• 1. BL01 XbaI/PstI
• 2. BL05 XbaI/PstI
• 3. BL09 XbaI/PstI
• 4 BL12 XbaI/PstI

Follow steps for gel electrophoresis.

• Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
• Fill gel flask with up to 60 ml of TA buffer.
• Create 1% gel by putting .6 grams of agarose into flask.
• Microwave agarose solution for 40 seconds
• Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
• When flask is taken out of microwave, make sure that the agarose is completed dissolved.
• Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
• Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
• Pour gel into tray.
• Wash the agarose gel flask.
• Run gel for 45 min at 100 V. Check gel under UV light.

Reagent	Volume	30 μL/lane, 1% agarose; Ladder
DNA (plasmid)	15.0 μL
XbaI	1.0 μL
PstI	1.0 μL
Green 10x FastDigest buffer	1.0 μL
dH2O	10 μL
	30 μL --> 37°C/ ~10 min.