Line 250: | Line 250: | ||
8. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading<div style="text-indent:60px;"> on the gels after the PCR.<div style="text-indent:60px;"> In a 2% agarose TAE gel the dye migrates with ~350 bp DNA, in 1% agarose TAE gel with ~600 bp <div style="text-indent:60px;">DNA fragments </div></div></div></div> | 8. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading<div style="text-indent:60px;"> on the gels after the PCR.<div style="text-indent:60px;"> In a 2% agarose TAE gel the dye migrates with ~350 bp DNA, in 1% agarose TAE gel with ~600 bp <div style="text-indent:60px;">DNA fragments </div></div></div></div> | ||
<br> <br> | <br> <br> | ||
− | <b>step by step for E.coli: </b | + | <b>3. Are you working with <i> A. E.coli </i> or B.yeast?</b> |
− | <div style="text-indent: | + | <br> <br> |
− | - Prepare masterplate | + | <div style="text-indent:20px;"> |
− | - Prepare a PCR master mix (always prepare at least 10 % more | + | <b>A.step by step for E.coli: </b> <br> |
− | + | <div style="text-indent:40px;"> | |
− | + | 1. Resuspend colonies </div> | |
− | Mix gently, avoid bubbles. < | + | <div style="text-indent:40px;"> |
− | - Aliquote the 22.0 μl of PCR master mix into each PCR tube. < | + | 2. Prepare masterplate </div> |
− | - Pick colony and put the toothpick or tip one into the Masterplate and then in the PCR tube/mix. < | + | <div style="text-indent:40px;"> |
− | < | + | 3. Prepare a PCR master mix (always prepare at least 10 % more) </div> |
− | Do not forget the negative control! < | + | <div style="text-indent:40px;"> |
− | < | + | 4. Mix gently, avoid bubbles. </div> |
− | + | <div style="text-indent:40px;"> | |
− | - Perform the PCR using Thermocycler as follow: <br> | + | 5. Aliquote the 22.0 μl of PCR master mix into each PCR tube. </div> |
+ | <div style="text-indent:40px;"> | ||
+ | 6. Pick colony and put the toothpick or tip one into the Masterplate and then in the PCR tube/mix. </div> | ||
+ | <div style="text-indent:40px;"> | ||
+ | 7. Do not forget the negative control! </div> | ||
+ | <div style="text-indent:40px;"> | ||
+ | 8. Close tube </div> | ||
+ | <div style="text-indent:40px;"> | ||
+ | 9. Perform the PCR using Thermocycler as follow: </div> | ||
+ | <br> | ||
<style type="text/css"> | <style type="text/css"> | ||
.tg {border-collapse:collapse;border-spacing:0;} | .tg {border-collapse:collapse;border-spacing:0;} | ||
Line 302: | Line 311: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <br> < | + | <br> |
− | + | <div style="text-indent:40px;"> | |
− | - Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round). < | + | 10. Store probes for short time on ice, for long at -20°C </div> |
+ | <div style="text-indent:40px;"> | ||
+ | 11. Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round). </div> | ||
<br> | <br> | ||
− | <b> | + | <b>B. Step by step for yeast </b> |
- 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 | - 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 | ||
resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for | resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for |
Revision as of 17:14, 29 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
Main sponsors: