Team:Imperial College London/Protocol
From 2010.igem.org
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Restriction Digests | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Restriction Digests | ||
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- | |Method: | + | |'''Method:''' |
#Determine the concentration of the DNA sample by running both the vector and insert ona 1% agarose gel and comparing the bands intensity with the ladder (concentration known). | #Determine the concentration of the DNA sample by running both the vector and insert ona 1% agarose gel and comparing the bands intensity with the ladder (concentration known). | ||
#Calculate how much solution is needed to obtain desired total amount of DNA for digestion. | #Calculate how much solution is needed to obtain desired total amount of DNA for digestion. | ||
#The volume of DNA solution can be no more than 70% of the total solution. Therefore calculate the total volume of digestion (probably around 20µl or 30µl). | #The volume of DNA solution can be no more than 70% of the total solution. Therefore calculate the total volume of digestion (probably around 20µl or 30µl). | ||
#Transfer the DNA, BSA, the appropriate buffer and ddH2O into a microcentrifuge tube. Finally, add the enzymes to the solution. N.B. The enzymes should be kept on ice before being added to the digestion. | #Transfer the DNA, BSA, the appropriate buffer and ddH2O into a microcentrifuge tube. Finally, add the enzymes to the solution. N.B. The enzymes should be kept on ice before being added to the digestion. | ||
- | #Incubate for 60-90min at 37°C. | + | #Incubate for 60-90min at 37°C. Put in the freezer or on ice immediately after to stop further digestion. Especially important for EcoRI and other enzymes with star activity. |
#Use gel electrophoresis to confirm correct digestion. | #Use gel electrophoresis to confirm correct digestion. | ||
#Gel purification can be used to obtain the desired digestion product from the gel. | #Gel purification can be used to obtain the desired digestion product from the gel. | ||
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'''Reaction mixtures:''' | '''Reaction mixtures:''' | ||
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- | The buffer depends on the restriction enzymes used | + | # 20µl reaction volume unless digesting large amounts of DNA (use 30µl) |
+ | # 4µl DNA (if from Midi-preps, use 8µl Mini-Prep DNA) | ||
+ | # 2µl Buffer * (1 in 10µl total volume) | ||
+ | # 2µl 10xBSA (1 in 10µl total volume) | ||
+ | # 1µl Enzyme 1 (use 1.5µl for 30µl digests) | ||
+ | # 1µl Enzyme 2 (most Bio-Brick REF assembly protocols require a second enzyme) (use 1.5µl for 30µl digests) | ||
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+ | The buffer depends on the restriction enzymes used. | ||
'''Prefix Insertion:''' | '''Prefix Insertion:''' | ||
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{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Ligations | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Ligations |
Revision as of 12:27, 14 October 2010
Restriction Digests | ||||||||||||||||||||||||||||||||||
Method:
Reaction mixtures:
The buffer depends on the restriction enzymes used. Prefix Insertion:
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Ligations |
A typical ligation reaction mixture is around 10 μl and contains
Insert mass (ng) = 6 x (Insert length (bp)/vector length (bp) x Vector mass (ng) Once the solution is made up, the tubes are vortexed and then spun down for around 10 seconds in a microcentrifuge. The ligation is done at 14°C in a water bath in the cold cabinet, and is left overnight. |
E. coli Transformations |
N.B. During pipetting the sides of the tube should not be touched to avoid contamination. Bubbles should be avoided because they can cause the cells stress.
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PCR |
PCR Reaction Mix
PCR programme
(-t°C optimal: 72°C for Taq // time optimal: 2-3kb/60 sec) (-t°C optimal:68°C for Pfu // time optimal: 1kb/15 sec)
We also used a positive control (other DNA to which the primers will definitely anneal) and a negative control (ddH20). The temperature cycle was as follows:
95°C for 30 seconds 62°C for 90 seconds 68 °C for 30 seconds
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Overnight Cultures |
Tubes containing 5ml of LB medium are inoculated with cells from one colony and then 5μl of antibiotic (for example chloramphenicol) is added. They are then left at 37°C overnight. |
SDS-PAGE |
Short for: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
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Catechol Assay |
* Catechol assay is performed in the plate reader on a 96 well plate
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Other Useful Information |
PCR purification
Used to purify DNA to remove primers, salts and enzymes. It can also be used to purify away small fragments from restriction digests, for example when cutting a vector open. We used the E.Z.N.A.® Cycle Pure Kit and protocol (Omega bio-tek) (ddH2O instead of Elusion Buffer used in last step). Gel purification We used the QIAquick® Gel Extraction Kit (250) and protocol (ddH2O instead of Elusion Buffer used in last step).
Midipreps The QIAGEN HiSpeed Plasmid Midi Kit and protocol was used. Agarose gels
Diluting Primers
Oligo annealing
Sequencing reaction mix
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