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<hr size="10" noshade></hr> | <hr size="10" noshade></hr> | ||
− | <p style="font-size: | + | <p style="font-size:10pt;"><sup>[1]</sup> |
http://parts.igem.org/Help:Protocols/3A_Assembly</p> | http://parts.igem.org/Help:Protocols/3A_Assembly</p> | ||
</div></div></div> | </div></div></div> | ||
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<div class="inner" style="display:none;"> | <div class="inner" style="display:none;"> | ||
− | + | <br> | |
<div alignt="justify"> | <div alignt="justify"> | ||
<b>1. Aim </b><br> | <b>1. Aim </b><br> | ||
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1. Is the insert DNA in the plasmid present or absent? </div> | 1. Is the insert DNA in the plasmid present or absent? </div> | ||
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 2. Much easier than to isolate and purify the vector </div> | + | 2. Much easier than to isolate and purify the vector. </div> |
− | + | <br> | |
<b> 2. Good to know before the start </b> <br> | <b> 2. Good to know before the start </b> <br> | ||
<div style="text-align: justify; margin-left:20px"> | <div style="text-align: justify; margin-left:20px"> | ||
− | 1. Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips | + | 1. Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips. |
<br> | <br> | ||
2. Include a no-template control and positive control in parallel. <br> | 2. Include a no-template control and positive control in parallel. <br> | ||
− | 3. Thaw and keep reagents on ice <br> | + | 3. Thaw and keep reagents on ice. <br> |
4. Mix well before use. <br> | 4. Mix well before use. <br> | ||
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5. The longer the amplicon, the longer the extension time: Use 15 sec/kb extension. <br> | 5. The longer the amplicon, the longer the extension time: Use 15 sec/kb extension. <br> | ||
− | 6. Use 90 sec extension for multiplexing <br> | + | 6. Use 90 sec extension for multiplexing. <br> |
7. Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing. <br> | 7. Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing. <br> | ||
− | 8. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR.In a 2% agarose TAE gel the dye migrates with ~350 bp DNA, in 1% agarose TAE gel with ~600 bp DNA fragments </div> | + | 8. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2 % agarose TAE gel the dye migrates with ~350 bp DNA, in 1 % agarose TAE gel with ~600 bp DNA fragments </div.> |
<br> <br> | <br> <br> | ||
<b>3. Are you working with <i> A. E.coli </i> or B.yeast?</b> | <b>3. Are you working with <i> A. E.coli </i> or B.yeast?</b> | ||
<br> <br> | <br> <br> | ||
<div style="text-indent:20px;"> | <div style="text-indent:20px;"> | ||
− | <b>A.step by step for E.coli: </b> <br> | + | <b>A.step by step for <i>E.coli</i>: </b> <br> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 1. Resuspend colonies </div> | + | 1. Resuspend colonies. </div> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 2. Prepare masterplate </div> | + | 2. Prepare masterplate. </div> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 3. Prepare a PCR master mix (always prepare at least 10 % more) </div> | + | 3. Prepare a PCR master mix (always prepare at least 10 % more). </div> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
4. Mix gently, avoid bubbles. </div> | 4. Mix gently, avoid bubbles. </div> | ||
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7. Do not forget the negative control! </div> | 7. Do not forget the negative control! </div> | ||
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 8. Close tube </div> | + | 8. Close tube. </div> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
9. Perform the PCR using Thermocycler as follow: </div> | 9. Perform the PCR using Thermocycler as follow: </div> | ||
Line 316: | Line 316: | ||
<br> | <br> | ||
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 10. Store probes for short time on ice, for long at - | + | 10. Store probes for short time on ice, for long at -20 °C. </div> |
<div style="text-indent:40px;"> | <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> | 11. Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round). </div> | ||
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<div style="text-indent:20px;"> | <div style="text-indent:20px;"> | ||
<b>B. Step by step for yeast: </b></div> | <b>B. Step by step for yeast: </b></div> | ||
− | <div style="text-align: justify; margin-left:40px"> | + | <div style="text-align: justify; margin-left:40px"><br> |
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 resuspended must just be 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 resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for | ||
≥ 5 min at 37 °C. <br> | ≥ 5 min at 37 °C. <br> | ||
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. <br> | 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. <br> | ||
− | 3. Prepare a PCR master mix (always prepare at least 10 % more | + | 3. Prepare a PCR master mix (always prepare at least 10 % more).<br> |
4. Aliquot 22.5 μl of PCR master mix into each PCR tube. <br> | 4. Aliquot 22.5 μl of PCR master mix into each PCR tube. <br> | ||
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7. Perform the PCR using the following cycling profile: </div><br> | 7. Perform the PCR using the following cycling profile: </div><br> | ||
− | + | <table> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <table | + | |
<tr> | <tr> | ||
− | <th | + | <th align="center"><b>Initial denaturation</b><br></th> |
− | <th | + | <th align="center">1 cycle<br></th> |
− | <th | + | <th align="center">95°C</th> |
− | <th | + | <th align="center">60s</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td | + | <td align="center"><b>Denaturation</b></td> |
− | <td | + | <td align="center">30-40 cycles<br></td> |
− | <td | + | <td align="center">95°C</td> |
− | <td | + | <td align="center">15s</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td | + | <td align="center"><b>Annealing</b></td> |
− | <td | + | <td align="center">30-40 cycles<br></td> |
− | <td | + | <td align="center">55-65°C</td> |
− | <td | + | <td align="center">15s</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td | + | <td align="center"><b>Extension</b></td> |
− | <td | + | <td align="center">30-40 cycles<br></td> |
− | <td | + | <td align="center">72°C</td> |
− | <td | + | <td align="center">15-90s</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td | + | <td align="center"><b>Final extension</b></td> |
− | <td | + | <td align="center">1 cycle<br></td> |
− | <td | + | <td align="center">72°C</td> |
− | <td | + | <td align="center">5 min<br></td> |
</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> |
<div style="text-indent:40px;"> | <div style="text-indent:40px;"> | ||
− | 9. Store probes for short time on ice, for long at -20°C </div> | + | 9. Store probes for short time on ice, for long at -20°C. </div> |
<br> <br> | <br> <br> | ||
Revision as of 14:48, 31 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
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