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

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3/18/22

Cutting inserts to be placed in mammalian vector

A) Get biobricks and enzymes from freezer.

PstI, XbaI

The following list of parts are:

  • 1. BL01
  • 2. BL05
  • 3. BL09
  • 4. BL12
Stock Digest

B)Follow digest procedure:

  • 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 Stock digest 3/18/2013 Stock digest 3/18/2013
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.


# Biobrick ng/µL
1 hPCD : mCh : SP1AB (BL01) 18.834
2 fshPCD : mCh : SP1AB (BL05) 14.471
3 flyPCD : mCh : SP1A (BL09) 7.302
4 hPCD : BL01 (BL12) 10.84



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