Koeris/Notebook/2007-1-19

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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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=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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===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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[[Image:2007-01-19_hipA-his6.tif|thumb|10% Rx load]]
 
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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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==Re-PCR and restriction digest==
 
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This time digested with Knp I and Xma I. Yield is [].
 
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Ligated into pZE21 digested with Kpn I and Xma I.
 
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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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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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