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2. Much easier than to isolate and purify the vector </div> | 2. Much easier than to isolate and purify the vector </div> | ||
<br> <br> | <br> <br> | ||
− | <b> 2. | + | <b> 2. Good to know before the start </b> <br> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
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<div style="text-indent:20px;"> | <div style="text-indent:20px;"> | ||
<b>B. Step by step for yeast </b> | <b>B. Step by step for yeast </b> | ||
− | + | <div style="margin-left:20px;"> | |
− | + | 1. If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH solution into each of a set of <div style="text-indent:40px;">appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks,<div style="text-indent:40px;"> transfer transformants to individual tubes/wells. The amount of cells resuspended must just be <div style="text-indent:40px;"> visible. Resuspend cells by pipetting or vortexing and incubate for | |
− | 1. If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks, transfer transformants to individual tubes/wells. The amount of cells | + | ≥ 5 min at 37 °C. </div></div></div></div> |
− | resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for | + | <div style="text-indent:40px;"> |
− | ≥ 5 min at 37 °C. </div></ | + | 2. If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of <div style="text-indent:60px;"> appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to <div style="text-indent:60px;"> be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at<div style="text-indent:60px;"> 37 °C. </div></div></div></div> |
− | < | + | <div style="text-indent:40px;"> |
− | 2. If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at 37 °C. </div></ | + | 3. Prepare a PCR master mix (always prepare at least 10% more, use the excel sheet to calculate)</div> |
− | < | + | |
− | 3. Prepare a PCR master mix (always prepare at least 10% more, use the excel sheet to calculate)</div | + | |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
4. Aliquot 22.5 μl of PCR master mix into each PCR tube. </div> | 4. Aliquot 22.5 μl of PCR master mix into each PCR tube. </div> | ||
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 5. Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the appropriate PCR tube. </div> | + | 5. Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the appropriate PCR<div style="text-indent:60px;"> tube. </div></div> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
6. Close the tubes </div> | 6. Close the tubes </div> | ||
− | <div style="text-indent:40px;"> | + | <div style="text-indent:40px;"><br> |
7. Perform the PCR using the following cycling profile: </div> | 7. Perform the PCR using the following cycling profile: </div> | ||
<style type="text/css"> | <style type="text/css"> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
− | + | ||
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
8. Load probes on the agarose gel </div> | 8. Load probes on the agarose gel </div> |
Revision as of 07:47, 30 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
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