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Team:EPF-Lausanne/Protocols/Klenow dsDNA synthesis
From 2011.igem.org
Klenow dsDNA synthesis
made on 5.07.2011
Materials
- 5’Comp Cy5 primer
- 5’Cy5-GTC ATA CCG CCG GAGG
- order from IDT at 1umole, HPLC purified
- suspend to 500uM
- Library primers:
- 5’ Linker - BINDING SITE – CCTCCGGCGGTATGAC
- 5’ Linker generally: CCC
- Klenow Fragment 3’-5’ exo-
- Order from NEB (Cat #: M0212L)
- dNTPs at 10mM each
Method
| 3 μL | dNTP |
| 3 μL | Buffer 2 (NEB Biolab) |
| 2 μL | Library primer (150μM) |
| 0.4 μL | 5’Comp Cy5 primer |
| 1 μL | Klenow 3’-5’ exo- |
| 20.6 µL | dH2O |
- 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:
- 37°C for 1 hour
- 75°C for 20 mins
- ramp down to 30°C at 0.1°C/sec
- hold at 4°C
- Alternatively in some cases use:
I will finish it later on ...
Alina
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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.
- To set up the 384-well plate with 6 dilutions load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19.
- 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.
- Transfer column 2 of the klenow plate into A7 and perform dilutions.
- Column3 -> A13, column 4 -> A19, Column 5->B1, col 6-> B7, col7->B13, col8 -> B19
- 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.
- The samples are now ready to be spotted.
