Koeris/Notebook/2007-1-19

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

10% Rx load

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