"
Team:UC Chile2/General Protocols
From 2012.igem.org
(Difference between revisions)
| Line 83: | Line 83: | ||
<h1>DNA assembly</h1> | <h1>DNA assembly</h1> | ||
</div> | </div> | ||
| - | < | + | <h2>Gibson Assembly</h2> |
| - | < | + | <h3>Design primers</h3> |
<p>The easiest way to design primers to obtain amplicons with the required overlaps (40bp final overlaps) is to make an <i>in sillico</i> design of the final construct</p> | <p>The easiest way to design primers to obtain amplicons with the required overlaps (40bp final overlaps) is to make an <i>in sillico</i> design of the final construct</p> | ||
<ul> | <ul> | ||
| Line 96: | Line 96: | ||
<li>Apply same principle in all joints</li> | <li>Apply same principle in all joints</li> | ||
</ul> | </ul> | ||
| - | < | + | <h3>Obtaining parts</h3> |
<ul> | <ul> | ||
<li>PCR parts using Phusion Polymerase datasheet indications. It is important to use a low ammount of template plasmid (10pg) as to reduce possibility of carry-over during band purification</li> | <li>PCR parts using Phusion Polymerase datasheet indications. It is important to use a low ammount of template plasmid (10pg) as to reduce possibility of carry-over during band purification</li> | ||
| Line 103: | Line 103: | ||
<li>Quantify purified DNA</li> | <li>Quantify purified DNA</li> | ||
</ul> | </ul> | ||
| - | < | + | <h3>Assembly reaction</h3> |
<p>The assembly reaction is composed of 9uL of 1.33X Gibson assembly master mix + 3uL of purified parts DNA</p> | <p>The assembly reaction is composed of 9uL of 1.33X Gibson assembly master mix + 3uL of purified parts DNA</p> | ||
<ul> | <ul> | ||
| Line 113: | Line 113: | ||
<li>Transform competent cells with 8uL of assembled DNA (that leaves 4uL to check in an agarose gel if the reaction fails)</li> | <li>Transform competent cells with 8uL of assembled DNA (that leaves 4uL to check in an agarose gel if the reaction fails)</li> | ||
</ul> | </ul> | ||
| - | < | + | <h3>Checking assembly</h3> |
<ul> | <ul> | ||
<li>Check transformant colonies by colony PCR using primers for whole insert</li> | <li>Check transformant colonies by colony PCR using primers for whole insert</li> | ||
Revision as of 04:32, 13 September 2012
Contents |
Growth Media
LB media
- 1% (w/v) tryptone
- 0.5% (w/v) yeast extract
- 86 mM NaCl
- 10g Tryptone
- 5g Yeast extract
- 5g NaCl
- 15g Agar -Optional-
- Adjust pH to 7.5 with NaOH 1M
Final concentrations:
Per liter:
SOB media
- 0.5% (w/v) yeast extract
- 2% (w/v) tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
- 5 g yeast extract
- 20 g tryptone
- 0.584 g NaCl
- 0.186 g KCl
- 2.4 g MgSO4
- 15g Agar -Optional-
- Adjust pH to 7.5 with NaOH 1M
Final concentrations:
Per liter:
Buffers
5X Gibson Assembly Isothermal Buffer
- 25% PEG MW 8000
- 500mM Tris HCl pH 7.5
- 50mM CaCl
- 50mM DTT
- 1mM dATP
- 1mM dTTP
- 1mM dGTP
- 1mM dCTP
- 5mM NAD+
- H20 nuclease free
- 0.5g PEG MW 8000
- 1.5mL Tris-HCl pH 7.5 1M
- 50uL CaCl 2M
- 100uL DTT 1M
- 20uL dATP 100mM
- 20uL dTTP 100mM
- 20uL dGTP 100mM
- 20uL dCTP 100mM
- 200uL NAD+ 200mM
- H20 nuclease free up to 2mL
Final Concentrations:
For a 2 mL stock (equivalent to 20 100uL 5X isothermal buffers)
Swivel for 30 minutes in a orbital oscillator for PEG to dissolve fully
Alicuot into 100uL stocks
Store at -80°C
1.33X Gibson Assembly Master Mix
- 100uL 5X Gibson Assembly Isothermal Buffer
- 6.25uL Phusion Polymerase 2 U/uL (cat N° F-350S) from Thermo Scientific
- 2uL Epicentre T5 Exonuclease 1U/uL (cat N° T5E4111K) from Illumina
- 50uL Taq DNA Ligase 2000 U/uL (cat N° M208S) from NEB
- 216.75uL of nuclease free H20
For a 375ul 1.33X Gibson Assembly Master Mix
It is important to dilute the T5 Exonuclease stock from 10U/uL to 1U/ul to measure the volume correctly
Alicuot 9uL in 0.2mL PCR tubes. This will yield about 42 reactions
DNA assembly
Gibson Assembly
Design primers
The easiest way to design primers to obtain amplicons with the required overlaps (40bp final overlaps) is to make an in sillico design of the final construct
- Design forward primer of right amplicon of joint in 5'->3' direction
- Calculate length of annealing part of primer as to reach a Tm of approximately 63°C
- Add 20 bp of overlap
- Design reverse primer of left amplicon of joint in 5'->3' direction
- To do this, select full joint and "reverse complement" it (be sure to be able to discriminate between right and left parts of joint)
- Calculate length of annealing part of primer as to reach a Tm of approximately 63°C
- Add 20 bp of overlap
- Apply same principle in all joints
Obtaining parts
- PCR parts using Phusion Polymerase datasheet indications. It is important to use a low ammount of template plasmid (10pg) as to reduce possibility of carry-over during band purification
- Run agarose gel electrophoresis on a adecuate gel (50bp to 200bp parts should be run on a 3% w/v agarose gel, larger parts should be run on a 1% w/v to 1.5% w/v agarose gel) until clear distinction of bands is achieved. It is recommended that gels should be exposed as little as possible to UV transilluminators as UV light damages DNA.
- Cut band and proceed with band purification. Elute in smallest volume as possible according to your kit specification
- Quantify purified DNA
Assembly reaction
The assembly reaction is composed of 9uL of 1.33X Gibson assembly master mix + 3uL of purified parts DNA
- The calculation of the ratio at which your DNA parts are should be in correspondence to the level of competence of your cells. We have found that with our cells (5*10^⁸ colonies/ug of pUC19 DNA) the following ratios work well
- Calculate the amount of pmoles of each purified DNA part using the following equation: pmoles of DNA = weigth in ng * 1000 (conversion factor from nano to pico) / (650 Daltons * base pair length of part)
- We have seen that using 0.01 picomoles of template (part with the selection resistance) and the rest of parts at 0.03 picomoles and above, yield a high ratio of true positive transformants and a decent number of colonies to check
- Thoroughly mix the 3uL of purified parts DNA with the 9uL of 1.33X Gibson assembly master mix in ice
- Directly incubate the reaction at 50°C for 1 hour
- Transform competent cells with 8uL of assembled DNA (that leaves 4uL to check in an agarose gel if the reaction fails)
Checking assembly
- Check transformant colonies by colony PCR using primers for whole insert
- Grow positive colonies in media with corresponding antibiotic
- Miniprep colonies and digest plasmid with EcoRI & PstI restriction enzymes
- If checking protocols validate until know, proceed by sequencing plasmid
