Difference between revisions of "Koeris/Notebook/2007-1-19"

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=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]<sub>6</sub>=
 
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
 
{| border="1"
 
! Primer !! Tube Label
 
|-
 
! A his6 2F&R 200pmol
 
| A1
 
|-
 
! A his6 2F&R 200pmol
 
| A2
 
|}
 
 
===Thermal profile===
 
#92 deg C - 5min
 
#92 deg C - 30s
 
#50 deg C - 45s
 
#72 deg C - 120s
 
#Cycle back to 2. 29X
 
#72 deg C - 10min
 
#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|thumb|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]<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
 

Latest revision as of 07:24, 13 February 2007