Team:EPF-Lausanne/Protocols/Klenow dsDNA synthesis

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(Created page with "{{:Team:EPF-Lausanne/Templates/Header|title=Klenow dsDNA synthesis}} made on 5.07.2011 == Materials == * 5’Comp Cy5 primer ** 5’Cy5-GTC ATA CCG CCG GAGG ** order from IDT a...")
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{{:Team:EPF-Lausanne/Templates/Header|title=Klenow dsDNA synthesis}}
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{{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Klenow dsDNA synthesis}}
made on 5.07.2011
made on 5.07.2011
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|20.6 µL
|20.6 µL
| dH2O
| dH2O
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|+Final volume
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|-
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|+30 µL
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|'''30 µL'''
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|'''Final volume'''
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<p>I will finish it later on ...</p>
 
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<p>Alina</p>
 
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<p> eom</p>
 
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Prepare a master mix without the library primer, and load 28μL into a 96 well plate. Add 2μL of Library primer to the 96 well plate. Cycle as follows:
 
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• 37°C for 1 hour
 
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• 75°C for 20 mins
 
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• ramp down to 30°C at 0.1°C/sec
 
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• hold at 4°C
 
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After the synthesis add 70μL of 0.5% BSA in dH2O to each well and transfer to a 384 well plate (flat bottom). Prepare dilutions as needed using 0.5% BSA dH2O.
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*Prepare a master mix without the library primer, and load 28μL into a 96 well plate.
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*Add 2μL of Library primer to the 96 well plate.
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*Cycle as follows:
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** 37°C for 1 hour
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** 75°C for 20 mins
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** ramp down to 30°C at 0.1°C/sec
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** hold at 4°C
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* After the synthesis add 70μL of 0.5% BSA in dH2O to each well and transfer to a 384 well plate (flat bottom). Prepare dilutions as needed using 0.5% BSA dH2O.
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To set up the 384 load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19.
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* To set up the 384-well  plate with 6 dilutions load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19.
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Transfer the column 1 of the klenow reaction (100uL total) to column 1 row A (A1) on the 384 well plate. Perform 5 dilutions of 70uL sample into the adjacent wells containing 30uL BSA dH2O. After the 5th dilution (in column 6) discard the remaining 70uL.
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* Transfer the column 1 of the klenow reaction (100uL total) to column 1 row A (A1) on the 384 well plate. Perform 5 dilutions of 70uL sample into the adjacent wells containing 30uL BSA dH2O. After the 5th dilution (in column 6) discard the remaining 70uL.
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Transfer column 2 of the klenow plate into A7 and perform dilutions.
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* Transfer column 2 of the klenow plate into A7 and perform dilutions.
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Column3 -> A13, column 4 -> A19, Column 5->B1, col 6-> B7,  col7->B13, col8 -> B19
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** Column3 -> A13, column 4 -> A19, Column 5->B1, col 6-> B7,  col7->B13, col8 -> B19
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You should have 30uL of target DNA in each well of the 384 plate now. Add 30uL of 0.5% BSA dH2O to each well.
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* You should have 30uL of target DNA in each well of the 384 plate now. Add 30uL of 0.5% BSA dH2O to each well.
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The samples are now ready to be spotted.
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* The samples are now ready to be spotted.

Latest revision as of 17:05, 21 September 2011

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