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| __TOC__
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| =DNA Fragment Purification from Acrylamide=
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| ==Solutions==
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| ===Crush and Soak Solution===
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| *500 mM NH4OAc 3.3 g NH4OAc
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| *0.1% SDS 0.1 g SDS
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| *0.1 mM EDTA 20 ml 500 mM EDTA
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| *up to 100 ml with Q
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| **store at room temperature
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| *3 M NaOAc pH 5.2
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| *24.6 g anhydrous sodium acetate
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| *pH to 5.2 with acetic acid and bring up to 100 ml with Q
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| **store at room temperature
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| ===Other Reagents===
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| *DMCS treated glass wool
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| *0.22 mm disposable micro tip filters (syringe type)
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| *blue tips with melted tips to serve as pestle for crushing acrylamide
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|
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| ==Procedure==
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| *Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band
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| *Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar.
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| *Add 1 ml crush and soak solution and incubate overnight at 37° C
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| *Spin in the microfuge for 10 minutes at 14,000 rpm
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| **Remove as much liquid as possible and '''KEEP IT'''
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| *Add another 500 microliters of crush and soak solution
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| *Repeat the spin and pool the recovered supernatant
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| *Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above)
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| *Spin as usual, wash and dry. Resuspend in 20 microliters TE (or Qiagen buffer EB)
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|
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| =Re-cloning of [His]<sub>6</sub>=
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| Due to lack of material, I could not recover the A plasmid and have to re-clone it.
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|
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| ==PCR set-up==
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| *100 ul total reaction volume
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| *1ul template DNA @ ~40ng/ul
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| *200pmol primer mix
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| *97ul Taq SuperMix
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| ===Pipetting scheme===
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| Labels in the respective field codes
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| {| border="1"
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| ! Primer !! Tube Label
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| |-
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| ! A his6 2F&R 200pmol
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| | A1
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| |-
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| ! A his6 2F&R 200pmol
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| | A2
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| |}
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|
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| ===Thermal profile===
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| #92 deg C - 5min
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| #92 deg C - 30s
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| #50 deg C - 45s
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| #72 deg C - 120s
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| #Cycle back to 2. 29X
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| #72 deg C - 10min
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| #4 deg C - indefinite
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| ==Gel image==
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| Loaded 10ul, i.e. 10% of Rx on the gel; 10ul 2-log ladder as well
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|
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| [[Image:2007-01-19_hipA-his6.tif|thumb|10% Rx load]]
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|
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| ==Restriction digest==
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| *Use Qiagen PCR cleanup kit to remove salts and enzymes
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| *Digest PCR amplicons with KpnI, Hind III - double digest for 2h
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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.
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| Used wrong enzyme - hipA has a cut site for Hind III
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|
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|
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| ==Re-PCR and restriction digest==
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| This time digested with Knp I and Xma I. Yield is [].
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|
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| Ligated into pZE21 digested with Kpn I and Xma I.
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|
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| ===Ligation of pZE21 & A [His]<sub>6</sub> construct===
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| Set up three different insert to backbone ratio reactions: Insert: backbone 1:1, 2:1 & 3:1
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|
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| Reaction set up
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| *T4 Ligase buffer [10X] 2ul
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| *Insert
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| *backbone
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| *T4 Ligase 1ul
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| *ddH20 to 20ul
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| *Total Rx volume 20ul
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