Revision as of 07:06, 24 September 2011

Protocol

Medium for drosophila

[http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]
Materials	Methods
water : 500 mL dry yeast : 20 g corn flour : 45 g glucose : 50 g agarose : 3.5~5 g propionic acid : 1.5 mL 10 % p-hydroxybenzoate in 70 % Eternol : 5 g	Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it. Stir corn flour and glucose with the remaining water. Stir 1 and 2, then autoclave it again. after autoclave, add propionic acid and 10 % p-hydroxybenzoate in 70 % Eternol into it.■

M9 medium

Materials	Methods
water : 1 L Na2HPO4: 6 g KH2PO4 : 3 g NaCl : 0.5 g NH4Cl : 1 g 100 mM MgSO4 : 10 ml 20 % glucose : 10 mL 10 mM CaCl2 : 10 ml 100 mM thiamine-HCl : 10 ml 20 % casamino acid : 10 ml	Stir Na2HPO4, KH2PO4, NaCl and NH4Cl with water. After autoclave, add 10 ml filter sterilized 100 mM MgSO4, 20 % glucose, 10 mM CaCl2, 100 mM thiamine-HCl. If you need, add 10 ml filter sterilized 20 % casamino acid.

LB medium

Materials	Methods
water : 1 L Tryptone: 10 g Yeast extract : 5 g NaCl : 10 g Agar : 15g	Stir Tryptone, Yeast extract and NaCl with water. If you make LB plates, add agar. Autoclave.

SOB medium

Materials	Methods
water : 100 mL Tryptone: 2 g Yeast extract : 0.5 g 5 M NaCl : 200 ul 3 M KCl : 84 ul 1 M MgSO4 : 1 ml 1 M MgCl2 : 1 ml	Stir Tryptone and Yeast extract with water. Add 200 ul 5 M NaCl and 84 ul 3 M KCl. After autoclave, add 1 ml filter sterilized 10 mM MgSO4 and 10 mM MgCl2.

SOC medium

Materials	Methods
SOB : 100 ml 2 M glucose: 1 ml	Add 2 M glucose to 100 ml SOB.

Buffer TB

Materials	Methods
water : 200 ml PIPES : 3 g CaCl2・2H2O : 0.22 g 3 M KCl : 8.315 ml KOH MnCl2・4H2O : 1.09 g	Stir PIPES and CaCl2・2H2O with 100 ml water. Add 8.315 ml 3 M KCl. Add KOH and adjust pH 6.8. Add MnCl2・4H2O. Add water up to 200 ml. Filter sterilize

DNS reaegnt

Materials	Methods
4.5% NaOH : 30 ml 1% DNS : 88 ml Rochelle salt : 25.5 g 10% NaOH : 2.2 ml Phenol : 1 g Water : 7.8 ml NaHCO3 : 0.69 g	Add 1% DNS 88ml and Rochelle salt 25.5 g to 4.5% NaOH 30 ml (A solution) Add Phenol 1 g and water 7.8 ml to 10% NaOH 2.2 ml (B solution) Add NaHCO3 6.9g to B solution 6.9 ml Add A solution 118ml to B solution 6.9 ml Leave 2 days Store in brown bottle

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 # Store in brown bottle
 |}
-PCR
-Standard PCR
-Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.
-Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.
-Mix the following.
-For use of KOD plus ver2:
-mM MgSO4
-µL
-mM dNTPs
-µL
-xBuffer for KOD plus ver.2
-µL
-Template DNA (5ng/µL)
-µL
-Primer Forward (10µM)
-.5µL
-Primer Reverse (10µM)
-.5µL
-KOD plus ver.2
-µL
-MilliQ
-µL
-Total
-µL
-For use of KOD FX:
-mM dNTPs
-µL
-xBuffer for KOD FX
-µL
-Template DNA
-µL
-Primer Forward (10µM)
-.5µL
-Primer Reverse (10µM)
-.5µL
-KOD FX
-µL
-MilliQ
-µL
-Total
-µL
-(If amplification is not succeeded, try at 4.5 or 6.0µL 25mM MgSO4.)
-Let stand for 2min at 94℃.
--40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
-At 15℃ forever.
-Agarose Gel Electrophoresis for confirmation.
-Screening PCR
-Mix the following (Do on PCR Bench).
-x PCR buffer (TAKARA)
-µL
-.5mM dNTP
-µL
-Primer-1 (10pmol/µL)
-µL
-Primer-2 (10pmol/µL)
-µL
-Ex Taq HS (TAKARA)
-.6µL
-MilliQ
-µL (to total 400µL)
-Dispense 25µL to 15 tubes.
-Pick a single colony and transfer it to each tubes.
-Suspend the colony.
-Let stand for 10min at 90℃.
-cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
-Let stand for 4min at 72℃.
-Add 5mL Loading Buffer to the tubes.
-Agalose Gel Electrophoresis for confirmation.
-Negative Control: Use nothing.
-Positive Control: Use a colony that will yield a product with this primers.
-Mutagenesis (Point mutation, Deletion, Insertion)
-Mix the following.
-xBuffer
-µL
-mM dNTP
-µL
-Primer Forward (10µM)
-.5µL
-Primer Reverse (10µM)
-.5µL
-Template Plasmid (50ng/µL)
-µL
-KOD plus ver.2
-µL
-MilliQ
-µL
-Total
-µL
-Prepare control: instead of KOD plus ver.2, add 1µL MilliQ.
-Let stand for 2min at 94℃.
-X cycles (1 cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
-Hold at 4℃.
-Take 25µL of the solutions into fresh tubes.
-Add 1µL DpnI (10U/µL).
-Let stand for 1h at 37℃.
-Agarose gel electrophoresis, using 5µL of the solution for confirmation.
-Mix the following.
-Sample
-µL
-Ligation high
-µL
-T4 Polynucleotide Kinase (5U/µL)
-µL
-MilliQ
-µL
-Total
-µL
-Let stand for 1h at 16℃.
-Transformation, using 10µL of the solution.
-PCR Purification
-Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN
-Add BufferPB about 5 times as much as the product of PCR.
-Apply the solution to the column.
-Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
-Add 750µL BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
-Centrifuge for 1min and discard the through.
-Centrifuge for additional 1min to remove residual buffer.
-Place the column in a clean tube.
-Add 10µL BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
-Centrifuge for 1min at 13000rpm.
-Discard the column.
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Team:Kyoto/Protocol

From 2011.igem.org

Revision as of 07:06, 24 September 2011

Contents

Protocol

Medium for drosophila

M9 medium

LB medium

SOB medium

SOC medium

Buffer TB

DNS reaegnt