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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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| + | *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: | ||
<p>I will finish it later on ...</p> | <p>I will finish it later on ...</p> | ||
<p>Alina</p> | <p>Alina</p> | ||
<p> eom</p> | <p> eom</p> | ||
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| - | To set up the 384 load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19. | + | *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 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. | + | *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 | + | **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. | + | *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. | + | *The samples are now ready to be spotted. |
Revision as of 15:43, 5 July 2011
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
eom
- 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.
