Koeris/Notebook/2007-1-19

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**Using Hind II might have been a mistake - a 200bp band showed up where it shouldn't have been -> check cut sites.
**Using Hind II might have been a mistake - a 200bp band showed up where it shouldn't have been -> check cut sites.
Used wrong enzyme - hipA has a cut site for Hind III
Used wrong enzyme - hipA has a cut site for Hind III
 +
 +
 +
==Re-PCR and restriction digest==
 +
This time digested with Knp I and Xma I. Yield is [].
 +
 +
Ligated into pZE21 digested with Kpn I and Xma I.
 +
 +
===Ligation of pZE21 & A [His]<sub>6</sub> construct===
 +
Set up three different insert to backbone ratio reactions: Insert: backbone 1:1, 2:1 & 3:1
 +
 +
Reaction set up
 +
*T4 Ligase buffer [10X] 2ul
 +
*Insert
 +
*backbone
 +
*T4 Ligase 1ul
 +
*ddH20 to 20ul
 +
*Total Rx volume 20ul

Revision as of 12:31, 24 January 2007

Contents

DNA Fragment Purification from Acrylamide

Solutions

Crush and Soak Solution

  • 500 mM NH4OAc 3.3 g NH4OAc
  • 0.1% SDS 0.1 g SDS
  • 0.1 mM EDTA 20 ml 500 mM EDTA
  • up to 100 ml with Q
    • store at room temperature
  • 3 M NaOAc pH 5.2
  • 24.6 g anhydrous sodium acetate
  • pH to 5.2 with acetic acid and bring up to 100 ml with Q
    • store at room temperature

Other Reagents

  • DMCS treated glass wool
  • 0.22 mm disposable micro tip filters (syringe type)
  • blue tips with melted tips to serve as pestle for crushing acrylamide

Procedure

  • Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band
  • Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar.
  • Add 1 ml crush and soak solution and incubate overnight at 37° C
  • Spin in the microfuge for 10 minutes at 14,000 rpm
    • Remove as much liquid as possible and KEEP IT
  • Add another 500 microliters of crush and soak solution
  • Repeat the spin and pool the recovered supernatant
  • Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above)
  • Spin as usual, wash and dry. Resuspend in 20 microliters TE (or Qiagen buffer EB)

Re-cloning of [His]6

Due to lack of material, I could not recover the A plasmid and have to re-clone it.

PCR set-up

  • 100 ul total reaction volume
  • 1ul template DNA @ ~40ng/ul
  • 200pmol primer mix
  • 97ul Taq SuperMix

Pipetting scheme

Labels in the respective field codes

Primer Tube Label
A his6 2F&R 200pmol A1
A his6 2F&R 200pmol A2

Thermal profile

  1. 92 deg C - 5min
  2. 92 deg C - 30s
  3. 50 deg C - 45s
  4. 72 deg C - 120s
  5. Cycle back to 2. 29X
  6. 72 deg C - 10min
  7. 4 deg C - indefinite

Gel image

Loaded 10ul, i.e. 10% of Rx on the gel; 10ul 2-log ladder as well

Image:2007-01-19 hipA-his6.tif

Restriction digest

  • Use Qiagen PCR cleanup kit to remove salts and enzymes
  • Digest PCR amplicons with KpnI, Hind III - double digest for 2h
    • Using Hind II might have been a mistake - a 200bp band showed up where it shouldn't have been -> check cut sites.

Used wrong enzyme - hipA has a cut site for Hind III


Re-PCR and restriction digest

This time digested with Knp I and Xma I. Yield is [].

Ligated into pZE21 digested with Kpn I and Xma I.

Ligation of pZE21 & A [His]6 construct

Set up three different insert to backbone ratio reactions: Insert: backbone 1:1, 2:1 & 3:1

Reaction set up

  • T4 Ligase buffer [10X] 2ul
  • Insert
  • backbone
  • T4 Ligase 1ul
  • ddH20 to 20ul
  • Total Rx volume 20ul
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