Haynes Lab:Notebook/Engineering PC-TFs/2012/02/15

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Electrophoesis Gel Preparation

  • 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.

First Stock Digest

Procedure

A) Get biobricks and enzymes from freezer.

SpeI, PstI, XbaI

The following list of biobricks are:

  • KAH01 hPCD
  • KAH03 flyPCD
  • KAH04 fshPCD
  • KAH204 mCh: SP1AB
  • KAH205 mCh: SP1A
  • KAH206 mCh: SP1B
  • KAH225 mCh: p65

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
Prep for Assembly
Prep for Assembly

Digest Order:

Biobrick Restriction Enzymes

  • 1. KAH01 SpeI/PstI
  • 2. KAH03 SpeI/PstI
  • 3. KAH04 SpeI/PstI
  • 4. KAH204 XbaI/PstI
  • 5. KAH205 XbaI/PstI
  • 6. KAH206 XbaI/PstI
  • 7. KAH225 XbaI/PstI


C) Put reactions into heat block for 10 minutes at 37°C

Note: Dr. Haynes completed 1, 6 and 7.


Photos

Digests 1-7
Gel Cut Out

Next Steps

  • DNA purification (QIAGEN) and then ligation.


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