Amide Synthesis with trimethyl borate
Synthesis of amide bonds with trimethyl borate as the coupling reagent. Used to produce polymers from p-aminobenzoic acid monomers.
- 2 equiv. P-aminobenzoic acid (pABA)
- 2 equiv. trimethyl borate
- Nonpolar/aprotic solvent (MeCN)
- Amide synthesis
- Add pABA (1 equiv) to MeCN (2 mL, 0.5 M)
- Add 1 equiv trimethyl borate to mixture. Stir at 80C (100C?) for 5-24 hours.
- Solid Extraction
- Dilute reaction mixture with CH2Cl2 or EtOAc (organic solvent) and H2O (polar solvent).
- Add MgSO4 (anhydrous) to remove H2O.
Annealing of Template DNA
Use this protocol to anneal complimentary single stranded oligonucleotides.
- Measurements per Tube:
- Forward Seq (200 µM) 10 µl
- Reverse Seq (200 µM) 10 µl
- 10x H Buffer 2 µl
- D.I. Water 28 µl
- Total Volume per Tube: 50 µl
- Final Concentration: 80 mM
- Anneal Strands
- Heat mixed tubes at 93°C for 1 min in thermal cycler
- Decrease heat of thermal cycler by 5°C every 2 min with small ∆T/sec in between steps
- When the temp falls more than 20°C below the Tm of the strands to anneal, change steps to decreasing 10°C every min
- Once at room temperature, hold samples at 4°C for 15 min, then store samples at -30°C
BCA Protein Assay
For determining concentration of protein using Nanodrop. summarized from http://www.nanodrop.com/Library/T085-NanoDrop-2000-2000c-BCA%20Protocol.pdf
- Working reagent (50A:1B)
- Pierce BCA Protein Assay Reagent A
- Pierce BCA Protein Assay Reagent B
- BSA/Albumin Standard (2mg/mL)
- PCR tubes/PCR plate
- Sample Prep
- Let all reagents/samples/standards equilibrate to RT.
- Prepare enough fresh working reagent (WR) for all standards and samples to be measured using a 50:1 ratio of the kit reagents A:B.
- If using high-range assay for 5 samples, you'll need approximately 1000 uL of WR total ==> So, you'll need about 1000 uL of A and 20 uL of B (ratio would be 1000:20).
- Add reagent volume to each PCR tube.
- Microassay (1:1 sample to WR) Add 10 uL of WR to each standards and sample tube/well.
- High range assay (1:20 sample to WR) Add 200 uL of WR to each standards and sample tube/well.
- Add 10 uL of standards or samples to the appropriate tube. Mix well by gentle vortexing.
- Incubate standard and sample tubes at:
- 37 C for 30 min OR 60 C for ~5 min. Cool to room temp after.
Cleaning Electrophoresis Gel
Cleaning electrophoresis gel with gel extraction kit (purple).
- 100% ethanol
- WN + WS buffers
- Gel Cleaning
- Prepare WN + WS buffers by adding appropriate amount of 100% ethanol as shown on bottle.
- Carefully cut out piece of gel containing fragment of interest under longwave UV.
- Add 300ul GEX for every 100mg gel collected to centrifuge tube and gel piece.
- Incubate at 55˚C for 5-10min until gel has dissolved. Can smash with a pipette tip.
- Place extraction column into collection tube and load 0.7ul mixture into column. Centrifuge at 5000rpm for 30s. Discard flow-through. Repeat until all mixture is used.
- Wash column with 0.5ml (500ul) of WN buffer by centrifuging at 5000rpm for 30s. Discard flow-through.
- Wash column with 0.5ml WS buffer by centrifuging for 60s at 5000rpm. Discard flow-through.
- Centrifuge column at 12000rpm for 3min to remove residual ethanol.
- Place column in new (storage) centrifuge tube. Add 15-30ul elution buffer onto center of membrane. Allow to stand for 2min.
- Centrifuge for 50s at 12000rpm to elute DNA. Store at -20˚C. Find concentration collected with Nanodrop.
Competent Cell Test Kit (iGEM)
Cell competency test on E.Coli strains with DNA kit provided by iGEM
- Purified DNA (BBa_J04450 RFP construct with plasmid backbone pSB1C3) at 0.5pg/ul, 5pg/ul, 10pg/ul, 20pg/ul, 50pg/ul (50ul aliquots)
- Competent cell aliquots
- Agar plates with chloramphenicol
- SOC media
- Spin down DNA tubes from Competent Cell Test Kit at 8,000-10,000rpm for 20-30 seconds
- Thaw competent cells on ice for 10 minutes. Label one 2.0ml microcentrifuge tube for each concentration and pre-chill by placing empty tubes on ice.
- Pipet 1uL of DNA into each microcentrifuge tube. Use a separate tube for each concentration.
- Pipet 50uL of competent cells into each tube, flicking tube gently to mix. Incubate on ice for 30 minutes. Preheat waterbath to 42˚C.
- Heat-shock cells by placing into waterbath for 1 minute.
- Immediately transfer tubes back to ice and incubate on ice for 5 minutes.
- Add 200uL of SOC per tube and incubate at 37˚C for 2 hours. Prepare and label agar plates.
- Pipet 20uL from each tube onto appropriate plate and evenly spread mixture across the plate. Do triplicates of each tube if possible to calculate an average colony yield.
- Incubate at 37˚C overnight or for 16 hours.
- Count number of colonies against a dark background, using an average cell colony count if done in triplicates.
- To measure competent cell efficiency: (colonies on plate) / ng of DNA plated x 1000ng/ug
- To measure ng of DNA plated: 1uL x concentration of DNA in original vial x (volume plated / total reaction volume)
- Competent cells have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA (cfu = colony-forming unit)
Cryostock Glycerol -80°C
Clonal bacterial freezer stocks are important for preserving plasmid constructs (adapted from Stanford BioE 44 F2015). Make a glycerol freezer stock from your bacterial culture as follows:
- 50%/40% glycerol
- Overnight culture of cells
- -80°C Freezer
- Making a clonal glycerol freezer stock
- Write down in your notebook the identifier and the plasmid strain you plan onstoring in the tube.
- Transfer 0.5 mL of 50% (or 40%) glycerol into each tube.
- Briefly vortex your bacterial culture to ensure that it is well mixed. Add 0.5 mLof the mixed culture to the appropriately labeled tube containing the glycerol
- Tightly cap the tube. Gently vortex the tube or invert multiple times to mix.
- Access the computer with the 2D barcode scanning hardware attached.Open the “BIOE44 F2014 glycerol stock” Excel file in the BIOE44 folder onthe computer.
- Record your unique identifier into the tube/notebook/spreadshseet and input the appropriate plasmid ID (e.g. pSB1C3, p70417, pComet, pX330-gRNA, orpJ1201-03c, etc.) into the plasmid ID column. Add into the notes column anynotes you deem appropriate. Remember to save the file when you are finished!
- Store the glycerol stock in a labeled box in the -80°C freezer.
Cryostock glycerol revival
Revive your frozen culture stocks
- Use a pipette tip to scrape off a little bit of the semi-thawed glycerol. Put tip with cells into media
- Put your glycerol stock back into the box in the -80°C freezer. Do not let it thaw completely! Thawing and freezing your stock repeatedly will kill your cells.
- Put inoculated culture into shaking incubator to let culture grow.
Written by Michael Becich, Julia Gross, Amy Weissenbach Adapted from Lan et al. 2012 (https://www.ideals.illinois.edu/bitstream/handle/2142/42441/Tian_Lan.pdf?sequence=1, http://www.hindawi.com/journals/jamc/2012/415697/)
- Streptavidin Beads
- 10mg/mL concentration (2 mL, 20mg total, 0.7-1.2X10^9 beads per mg)
- Random Library
- ATA CCA GCT TAT TCA ATT NNN NNN NNN NTG AGG CTC GAT CNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNA GAT AGT AAG TGC AAT CT
- Biotinylated Capture Oligo's (25 nmole)
- /5Biosg/TA CCG CAA AAA AAA AGA TCG AGC CTC A
- Use 750 pmol oligo for 150uL (1.5mg) beads, as rounds go on should consider cutting down
- PCR Primers (Forward, Reverse, and Elongated Reverse) (25 nmole)
- FWD: ATACCAGCTTATTCAATT
- RVS: GATTGCACTTACTATCT
- RVS_plus_spacer: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA/iSp18/GATTGCACTTACTATCT
- Selection Buffer (SB) - make 200 mL
- 100 mM NaCl - 0.005mol, about 0.3g/50mL
- 20 mM Tris-HCl pH7.6
- 2 mM MgCl2 -- 0.095g/50mL
- 5 mM KCl -- .0186 g/ 50mL
- 1 mM CaCl2 --- 0.005549g / 50mL
- Binding & Washing Buffer (B&W) - make 120 mL
- 10 mM Tris-HCl pH7.5
- 1 mM EDTA
- 2 M NaCl
- TE Buffer
- 10mM Tris-HCl pH 7.4
- 1 mM EDTA
- TBE Buffer
- 90 mM Tris-Hcl
- 90 mM boric acid
- 2 mM EDTA