Haynes Lab:Notebook/Engineering PC-TFs/2013/10/11

From OpenWetWare

< Haynes Lab:Notebook | Engineering PC-TFs | 2013 | 10(Difference between revisions)
Jump to: navigation, search
(Summary)
Current revision (00:18, 12 October 2013) (view source)
(October 11, 2013)
 
(One intermediate revision not shown.)
Line 7: Line 7:
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==October 11, 2013==
==October 11, 2013==
-
* Follow steps for gel electrophoresis.
+
'''Restriction Digest'''
 +
 
 +
''Gel Electrophoresis''
 +
 
*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
*  Fill gel flask with up to 60 ml of TA buffer.
*  Fill gel flask with up to 60 ml of TA buffer.
Line 38: Line 41:
| &nbsp; || 30 μL --> 37°C/ ~10 min.
| &nbsp; || 30 μL --> 37°C/ ~10 min.
|}
|}
 +
 +
''Zymoclean<sup>TM</sup> Gel DNA Recovery Kit''
 +
 +
*Add 3 volumes of ADB Buffer to each volume of gel (600 μL).
 +
*Incubate at 55°C for 10 minutes.
 +
*Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through.
 +
*Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step.
 +
*Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH<sub>2</sub>O to elute DNA
{|border="1" cellpadding="5" cellspacing="0" align="left"
{|border="1" cellpadding="5" cellspacing="0" align="left"
|-
|-
! scope="col" style="background:#efefef;" | #
! scope="col" style="background:#efefef;" | #
-
! scope="col" style="background:#efefef;" | Sequence
+
! scope="col" style="background:#efefef;" | Part
! scope="col" style="background:#efefef;" | ng/µL
! scope="col" style="background:#efefef;" | ng/µL
|-
|-
|1
|1
|CMV
|CMV
-
| -1.17
+
|0.851
|-
|-
|2
|2
|CMV
|CMV
-
|2.767
+
|15.535
|}
|}

Current revision



Main project page
Previous entry      Next entry

October 11, 2013

Restriction Digest

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 CMV Stock digest 10/11/2013 CMV Stock Digest 10/11/2013
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
XbaI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 3.0 μL
dH2O 5 μL
  30 μL --> 37°C/ ~10 min.

ZymocleanTM Gel DNA Recovery Kit

  • Add 3 volumes of ADB Buffer to each volume of gel (600 μL).
  • Incubate at 55°C for 10 minutes.
  • Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through.
  • Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step.
  • Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH2O to elute DNA
# Part ng/µL
1 CMV 0.851
2 CMV 15.535



Personal tools