Difference between revisions of "Team:Paris Saclay/Experiments"
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{{Paris_Saclay|submenu={{Team:Paris_Saclay/submenu_labwork}}|labwork=active}} | {{Paris_Saclay|submenu={{Team:Paris_Saclay/submenu_labwork}}|labwork=active}} | ||
<html> | <html> | ||
+ | <h1>Labwork</h1> | ||
+ | <h2 id="protocols">Protocols</h2> | ||
− | < | + | <h3>Gibson assembly</h3> |
− | < | + | <h4>1. <u>Principle:</u> One-step isothermal in vitro recombination</h4> |
− | < | + | <p>Two adjacent DNA fragments sharing <b>terminal sequence 20 bp</b> overlaps were joined into a covalently sealed molecule in a one-step isothermal reaction.</p> |
+ | <p><b>T5 exonuclease</b> removed nucleotides <b>from the 5'</b> ends of doublestranded DNA molecules, complementary single-stranded DNA overhangs annealed.</p | ||
+ | <p><b>Phusion DNA polymerase</b> filled the gaps and Taq DNA ligase sealed the nicks.</br>T5 exonuclease is heat-labile and is inactivated during the 50°C incubation.<p> | ||
+ | <p>Incubation at 50°C during 1 hour --> the product can be used to transform into <i>E. coli</i> directly.</p> | ||
+ | |||
+ | <h4>2. Design primer</h4> | ||
+ | <p>Primer overlap at least 20 bp with the adjacent fragments (plasmid, fragments)</p> | ||
+ | |||
+ | <p>Order primer: IDT Company</br>Scale: 25nm DNA oligo</br>Normalization: None</br>Purification: standard desalting</p> | ||
+ | <p>The Tm is defined as the temperature in degrees Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands (the melting temperature).</br><b>Tm of overlap part must be higher than 50°C</b>. | ||
+ | |||
+ | <h4>3. PCR amplification</h4> | ||
+ | <p>Using enzyme Phusion High-Fidelity DNA polymerase (the most accurate thermostable polymerase available).</p> | ||
+ | |||
+ | <h4>4. Clean PCR, plasmid</h4> | ||
<ul> | <ul> | ||
− | <li> | + | <li><b>For DNA fragments</b>: Gel purify if it has an unspecific band or purify PCR product by kit. Estimate the concentration.</li> |
− | < | + | <li><b>For plasmid</b>: after PCR, digest your PCR product by enzyme DpnI (recognize and cut methylated DNA, allow to remove remaining plasmid template, and does not cleave PCR-amplified DNA). Then, purify your PCR plasmid digestion and estimates the concentration.</li> |
− | <li> | + | |
</ul> | </ul> | ||
− | <h4> | + | <h4>5. Gibson amplification</h4> |
<ul> | <ul> | ||
− | <li> | + | <li>Add the combined fragments (5 µl) to one Isothermal Assembly reaction aliquot (15 µl) and mix by pipetting (20 µl total).</li> |
− | <li> | + | <li>Not exceeding a total volume of 20 µl. If required, bring to 5 µl with ddH2O. <b>100 ng of plasmid is recommended</b>.</li> |
− | <li>< | + | <li>Incubate the mix at 50°C for 60 min.</li> |
+ | <li>Transform E. coli DH5α with 10 µl of assembly mix.</li> | ||
</ul> | </ul> | ||
− | <h4> | + | <h4>6. Transformation in <i>E. coli</i></h4> |
− | <p> | + | <p>Negative control: transform <i>E. coli</i> with the plasmid PCR product digested with DpnI --> know the background.</p> |
+ | |||
+ | |||
+ | <h3>Isothermal assembly</h3> | ||
+ | <p>Isothermal Assembly works by combining a cocktail of exonuclease, polymerase, and ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT overlap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50C.</p> | ||
+ | <p>To perform isothermal assembly:</p> | ||
+ | <ol> | ||
+ | <li>PCR up your fragments of choice, and purify. Also gel purify the cut vector.</li> | ||
+ | <li>Not exceeding a total volume of 5 µl, in a PCR tube, combine fragments at equal molecular ratio. If required, bring to 5 µl with ddH2O. Use approx. 100 ng of plasmid backbone.</li> | ||
+ | <li>Add the combined fragments (5 µl) to 1 Isothermal Assembly reaction aliquot (15 µl) and mix by pipetting (20 µl total).</li> | ||
+ | <li>Place mix at 50°C for 60 min.</li> | ||
+ | <li>Transform with 10 µl of assembly mix.</li> | ||
+ | </ol> | ||
+ | <p><b>References:</b></p> | ||
+ | <p>Gibson et al (2009) Nature Methods 6(5):343-345.</p> | ||
+ | <p><b>Making Isothermal Assembly Aliquots</b></p> | ||
+ | <p>5x isothermal assembly reaction buffer (assemble on ice):</p> | ||
+ | <table class="wikitable"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td width="240"><strong>From the paper:</strong></td> | ||
+ | <td width="200"><strong>Actually added:</strong></td> | ||
+ | </tr> | ||
+ | <tr><td>3 mL 1M Tris-HCl pH 7.5</td><td>3 mL 1M Tris-HCl pH 7.5</td></tr> | ||
+ | <tr><td>150 µL 2M MgCl<sub>2</sub></td><td>300 µL 1M MgCl<sub>2</sub></td></tr> | ||
+ | <tr> | ||
+ | <td>60 µL 100 mM dGTP</td> | ||
+ | <td>600 µL 10 mM each dNTP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>60 µL 100 mM dCTP</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>60 µL 100 mM dTTP</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>60 µL 100 mM dATP</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>300 µL 1M DTT/td> | ||
+ | <td>300 µL 1M DTT</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1.5 g PEG-8000</td> | ||
+ | <td>1.5 g PEG-8000</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>300 µL 100 mM NAD</td> | ||
+ | <td>20 mg NAD</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH<sub>2</sub>O to 6 ml</td> | ||
+ | <td>ddH<sub>2</sub>O to 6 ml</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>Prepare 320 µL aliquots (18) and freeze all but one. * Label these “5X isotherm buffer”</br>To the one remaining (320 µL), add:</p> | ||
+ | <table class="wikitable"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td width="90"> | ||
+ | <p> | ||
+ | 1.2 µL | ||
+ | </p> | ||
+ | </td> | ||
+ | <td width="215"> | ||
+ | <p> | ||
+ | T5 Exonuclease | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | 20 µL | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | Phusion polymerase | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | 160 µL | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | Taq ligase | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | 700 µL | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | ddH<sub>2</sub>O | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>Prepare 15 µL aliquots (~80) on ice in PCR tubes and store at -20C. These should be good for up to a year.</p> | ||
+ | <p> | ||
+ | <a name="Shopping_List"></a> | ||
+ | <strong>Shopping List </strong> | ||
+ | </p> | ||
+ | <p> | ||
+ | Buffer | ||
+ | </p> | ||
+ | <table class="wikitable"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td width="300"> | ||
+ | <p> | ||
+ | <strong>Item </strong> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td width="120"> | ||
+ | <p> | ||
+ | <strong>Vendor </strong> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td width="120"> | ||
+ | <p> | ||
+ | <strong>Ref </strong> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | 1M Tris-HCl pH 7.5 | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | Sigma | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | T2319 | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | Magnesium Chloride, 1.00 +/- 0.01M Solution | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | Affymetrix / USB | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | 78641 10 x 1 ml | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | Nicotinamide adenine dinucleotide (NAD) | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | VWR | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | A1124,0005 | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | DTT, molecular biology grade | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | FERMENTAS | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | R0861 | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | Polyethylene Glycol 8000, Powder | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | USB / Affymetrix | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | 19966 | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | dNTP Mix, 10mM each | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | Fermentas | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | R0192 1 ml | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p> | ||
+ | Enzymes: | ||
+ | </p> | ||
+ | <table class="wikitable"> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td width="300"> | ||
+ | <p> | ||
+ | T5 Exonuclease | ||
+ | </p> | ||
+ | </td> | ||
+ | <td width="120"> | ||
+ | <p> | ||
+ | NEB | ||
+ | </p> | ||
+ | </td> | ||
+ | <td width="120"> | ||
+ | <p> | ||
+ | M0363S | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | Taq DNA Ligase | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | NEB | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | M0208L | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p> | ||
+ | Phusion™ High-Fidelity DNA Polymerase | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | Thermoscientific | ||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | F530-S | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
</html> | </html> | ||
{{Team:Paris_Saclay/footer}} | {{Team:Paris_Saclay/footer}} |
Revision as of 09:27, 15 September 2015
Labwork
Protocols
Gibson assembly
1. Principle: One-step isothermal in vitro recombination
Two adjacent DNA fragments sharing terminal sequence 20 bp overlaps were joined into a covalently sealed molecule in a one-step isothermal reaction.
T5 exonuclease removed nucleotides from the 5' ends of doublestranded DNA molecules, complementary single-stranded DNA overhangs annealed.
Phusion DNA polymerase filled the gaps and Taq DNA ligase sealed the nicks.T5 exonuclease is heat-labile and is inactivated during the 50°C incubation.
Incubation at 50°C during 1 hour --> the product can be used to transform into E. coli directly.
2. Design primer
Primer overlap at least 20 bp with the adjacent fragments (plasmid, fragments)
Order primer: IDT CompanyScale: 25nm DNA oligoNormalization: NonePurification: standard desalting
The Tm is defined as the temperature in degrees Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands (the melting temperature).Tm of overlap part must be higher than 50°C.
3. PCR amplification
Using enzyme Phusion High-Fidelity DNA polymerase (the most accurate thermostable polymerase available).
4. Clean PCR, plasmid
- For DNA fragments: Gel purify if it has an unspecific band or purify PCR product by kit. Estimate the concentration.
- For plasmid: after PCR, digest your PCR product by enzyme DpnI (recognize and cut methylated DNA, allow to remove remaining plasmid template, and does not cleave PCR-amplified DNA). Then, purify your PCR plasmid digestion and estimates the concentration.
5. Gibson amplification
- Add the combined fragments (5 µl) to one Isothermal Assembly reaction aliquot (15 µl) and mix by pipetting (20 µl total).
- Not exceeding a total volume of 20 µl. If required, bring to 5 µl with ddH2O. 100 ng of plasmid is recommended.
- Incubate the mix at 50°C for 60 min.
- Transform E. coli DH5α with 10 µl of assembly mix.
6. Transformation in E. coli
Negative control: transform E. coli with the plasmid PCR product digested with DpnI --> know the background.
Isothermal assembly
Isothermal Assembly works by combining a cocktail of exonuclease, polymerase, and ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT overlap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50C.
To perform isothermal assembly:
- PCR up your fragments of choice, and purify. Also gel purify the cut vector.
- Not exceeding a total volume of 5 µl, in a PCR tube, combine fragments at equal molecular ratio. If required, bring to 5 µl with ddH2O. Use approx. 100 ng of plasmid backbone.
- Add the combined fragments (5 µl) to 1 Isothermal Assembly reaction aliquot (15 µl) and mix by pipetting (20 µl total).
- Place mix at 50°C for 60 min.
- Transform with 10 µl of assembly mix.
References:
Gibson et al (2009) Nature Methods 6(5):343-345.
Making Isothermal Assembly Aliquots
5x isothermal assembly reaction buffer (assemble on ice):
From the paper: | Actually added: |
3 mL 1M Tris-HCl pH 7.5 | 3 mL 1M Tris-HCl pH 7.5 |
150 µL 2M MgCl2 | 300 µL 1M MgCl2 |
60 µL 100 mM dGTP | 600 µL 10 mM each dNTP |
60 µL 100 mM dCTP | |
60 µL 100 mM dTTP | |
60 µL 100 mM dATP | |
300 µL 1M DTT/td> | 300 µL 1M DTT |
1.5 g PEG-8000 | 1.5 g PEG-8000 |
300 µL 100 mM NAD | 20 mg NAD |
ddH2O to 6 ml | ddH2O to 6 ml |
Prepare 320 µL aliquots (18) and freeze all but one. * Label these “5X isotherm buffer”To the one remaining (320 µL), add:
1.2 µL |
T5 Exonuclease |
20 µL |
Phusion polymerase |
160 µL |
Taq ligase |
700 µL |
ddH2O |
Prepare 15 µL aliquots (~80) on ice in PCR tubes and store at -20C. These should be good for up to a year.
Buffer
Item |
Vendor |
Ref |
1M Tris-HCl pH 7.5 |
Sigma |
T2319 |
Magnesium Chloride, 1.00 +/- 0.01M Solution |
Affymetrix / USB |
78641 10 x 1 ml |
Nicotinamide adenine dinucleotide (NAD) |
VWR |
A1124,0005 |
DTT, molecular biology grade |
FERMENTAS |
R0861 |
Polyethylene Glycol 8000, Powder |
USB / Affymetrix |
19966 |
dNTP Mix, 10mM each |
Fermentas |
R0192 1 ml |
Enzymes:
T5 Exonuclease |
NEB |
M0363S |
Taq DNA Ligase |
NEB |
M0208L |
Phusion™ High-Fidelity DNA Polymerase |
Thermoscientific |
F530-S |