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Team:UC Davis/Notebook/Protocols
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| - | <br>PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension: As an important step in our project we needed to use a technique called PCR SOEing which is used to attach DNA fragments together without the many steps of digesting and ligating. Our two enzymes that break down ethylene glycol, glycoaldehyde reductase and glycoaldehyde dehydrogenase, needed to have the Eco, Xba, Spe or Pst restriction sites removed in order to biobrick the enzyme. So we had to look at the sequence of both enzymes and determine if they had any of the restriction site sequences. What we found was that glycoaldehyde reductase had one Pst restriction site and glcoaldehyde dehydrogenase had two Pst sites. | + | <br>PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension: As an important step in our project we needed to use a technique called PCR SOEing which is used to attach DNA fragments together without the many steps of digesting and ligating. Our two enzymes that break down ethylene glycol, glycoaldehyde reductase and glycoaldehyde dehydrogenase, needed to have the Eco, Xba, Spe or Pst restriction sites removed in order to biobrick the enzyme. So we had to look at the sequence of both enzymes and determine if they had any of the restriction site sequences. What we found was that glycoaldehyde reductase had one Pst restriction site and glcoaldehyde dehydrogenase had two Pst sites. <br> |
How SOEing works: in order to do SOEing, forward and reverse primers must be designed for where you want to attach and the whole enzyme as well. This is done by PCR reactions, and the ways we assay if the DNA fragments annealed is by doing a digest with the coordinating restriction enzyme. | How SOEing works: in order to do SOEing, forward and reverse primers must be designed for where you want to attach and the whole enzyme as well. This is done by PCR reactions, and the ways we assay if the DNA fragments annealed is by doing a digest with the coordinating restriction enzyme. | ||
Revision as of 17:25, 22 August 2012
Protocols
•Add 20ul sterile H2O to desired well in distribution plate.
•Incubate at room temperature for ~10 minutes.
•Transfer resuspension to microcentrifuge tube.
Materials
•Competent Cells•DNA template
•800 ul LB
•LB+Antibiotic Plates
Procedure
•Thaw competent cells on ice.•Transfer 50 uL of competent cells to chilled falcon tubes.
•Add 1 uL of template to cells (2.5 uL if dilute).
•Incubate on ice for 30 minutes.
•Heat schock in 42 °C water bath for 90 seconds.
•Immediately place back onto ice for 2 minutes.
•Add 800 uL of LB to each tube.
•Incubate at 37 °C for 1 hour.
•Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic.
•Incubate overnight at 37 °C.
Materials
•22 uL dH20•1 uL BSA
•5 ul Buffer
•20 uL Template
•1 uL Enzyme 1
•1 uL Enzyme 2
Procedure
•Mix reactants, adding enzyme last.•Place at 37 C for 3-5 hours.
•If not purifying or running on a gel immediately, increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
•Run on a gel and extract product.
Materials
•10 uL Q Solution•5 uL 10x Buffer
•1.25 uL 10mM dNTPs
•1 uL Template
•1 uL Forward Primer
•1 uL Reverse Primer
•0.3 uL Taq
•0.15 uL PFU
•30 uL dH20
Procedure
•Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.•Run in thermal cycler.
PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension: As an important step in our project we needed to use a technique called PCR SOEing which is used to attach DNA fragments together without the many steps of digesting and ligating. Our two enzymes that break down ethylene glycol, glycoaldehyde reductase and glycoaldehyde dehydrogenase, needed to have the Eco, Xba, Spe or Pst restriction sites removed in order to biobrick the enzyme. So we had to look at the sequence of both enzymes and determine if they had any of the restriction site sequences. What we found was that glycoaldehyde reductase had one Pst restriction site and glcoaldehyde dehydrogenase had two Pst sites.
How SOEing works: in order to do SOEing, forward and reverse primers must be designed for where you want to attach and the whole enzyme as well. This is done by PCR reactions, and the ways we assay if the DNA fragments annealed is by doing a digest with the coordinating restriction enzyme. The protocol is coming soon!
Materials
•Digested Vector•Digested Insert
•Water
•T4 DNA Ligase
•T4 DNA Ligase Buffer
Procedure
•Mix these materials in the amounts determined by the reaction volume calculator here.Materials
•34 mM NaH2PO4•64 mM K2HPO4
•20 mM (NH4)2SO4
•1 µM FeSO4
•0.1 mM MgSO4
•10 µM CaCl2
•30 mM Ethylene Glycol
•30 mM Glycolate
•0.5% wt/vol Casein Acid Hydrolysate
•1.5% wt/vol Agar (if making solid media)
Procedure
•Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.•Mix together NaH2PO4, K2HPO4, (NH4)2SO4, FeSO4, MgSO4, and CaCl2.
•Titrate to pH 7 with HCl.
•Add the glycolate and casein acid hydrolysate.
•Mix in the ethylene glycol in a fume hood.
•If making solid media, also mix in agar.
•Autoclave on an appropriate cycle.
Materials
• Luria Broth Media• Ethylene Glycol
• Tecan M200 Pro
• E. coli
Procedure
• Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.1. Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
2. Incubate at 37°C overnight on a shaker at 150 rpm.
3. From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
4. Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
5. Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
6. Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
7. With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.
8. Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
9. Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.
10. Aliquot the remaining dilutions as the diagram below depicts.
11. Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
12. Follow the steps of your Tecan machine appropriate to the specific bacterial strain.
