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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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| | | | |
| - | ==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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| - | ==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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