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

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=DNA Fragment Purification from Acrylamide=
===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
*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
*T4 Ligase 1ul
*ddH20 to 20ul
*Total Rx volume 20ul

Latest revision as of 07:24, 13 February 2007