SBB09Ntbk-Patrick Harrigan

February 20, 2009
I made 100uM dillutions from my oligos and spun them down. For a wobble reaction for KILLR, I added to a PCR tube:

29 uL water 5 uL Expand 10x Buffer 2 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) 5 uL Oligo Oph22F (100uM) 5 uL Oligo Oph23R (100uM) 0.75 uL Expand Polymerase 1

For my PCR Product traA, First I diluted my Oligos 9uL Water 1uL 100uM Oph24F/R

Then I set up the following reaction in a PCR tube 24uL ddH2O 3.3uL 10x Expand Buffer "2" 3.3uL dNTPs (2mM in each) 1uL Oligo 1, 10uM 1uL Oligo 2, 10uM 0.5uL Expand polymerase "1" 0.5uL Template DNA

For KILLR I ran the thermocycler on the wobble program. For traA, the thermocycler was run at 4k55

traA
I added 2 uL from my PCR reaction and a 0.25 uL of loading dye and ran an analytic gel: My product is in the fith lane, I am not sure why there are two fragments. I cleaned up my traA PCR reaction using the following protocol: I did not dry for long enough and may have gotten insufficient elution. I eluted with 50uL instead of 33 to try to make sure all DNA was eluted.
 * 1) Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
 * 2) Transfer into the Zymo column (small clear guys)
 * 3) Add 500uL of Ethanol and pipette up and down to mix
 * 4) spin through, discard waste.
 * 5) Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
 * 6) spin through, discard waste.
 * 7) Add 200 uL of PE or Zymo Wash buffer
 * 8) spin through, discard waste.
 * 9) spin for 90 seconds, full speed to dry.
 * 10) elute with water into a fresh Eppendorf tube

KILLR
I cleaned up my pcr product: For my wobble products, I digested the entire extension reaction-worth of DNA. 50uL eluted DNA 5.7uL NEB Buffer 2 1uL EcoRI 1uL BamHI
 * 1) Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
 * 2) Transfer into the Zymo column (small clear guys)
 * 3) Add 500uL of Ethanol and pipette up and down to mix
 * 4) spin through, discard waste.
 * 5) Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
 * 6) spin through, discard waste.
 * 7) Add 200 uL of PE or Zymo Wash buffer
 * 8) spin through, discard waste.
 * 9) spin for 90 seconds, full speed to dry.
 * I Set up the following reaction in a PCR tube:
 * I mixed thoroughly by slamming the tube upside down on the table, and then gave it a quick spin to move the liquid to the bottom of the tube.
 * I incubated the reaction at 37 degrees on the thermocycler

After digesting, I did a small Zymo cleanup:
 * 1) Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
 * 2) Transfer into the Zymo column (small clear guys)
 * 3) Add 500uL of Ethanol and pipette up and down to mix
 * 4) spin through, discard waste.
 * 5) Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
 * 6) spin through, discard waste.
 * 7) Add 200 uL of PE or Zymo Wash buffer
 * 8) spin through, discard waste.
 * 9) spin for 90 seconds, full speed to dry.

traA
I digested the cleanup of my PCR reaction: 8uL of eluted PCR product 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI And Incubated for 1 hour at 37 degrees celsius

I then did a zymo cleanup eluting into 50uL of water:
 * 1) Add 180 uL of Zymo ADB buffer (brown bottle) to a 50uL reaction.
 * 2) Transfer into the Zymo column (small clear guys)
 * 3) spin through, discard waste.
 * 4) Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
 * 5) spin through, discard waste.
 * 6) Add 200 uL of PE or Zymo Wash buffer
 * 7) spin through, discard waste.
 * 8) spin for 90 seconds, full speed to dry.
 * 9) elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

KILLR
I ligated my cleanup digestion: 6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase
 * Pound upside down on the bench to mix
 * Give it a quick spin to send it back to the bottom of the tube
 * Incubate on the benchtop for 30min
 * Put on ice and proceed to the transformation

I did a heat-shock transformation and plated on KA plates:
 * 1) Thaw a 200 uL aliquot of cells on ice
 * 2) Add 50 uL of water
 * 3) Add 30 uL of KCM salts
 * 4) Put your ligation mixture on ice, let it cool a minute or two
 * 5) Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
 * 6) Let sit on ice for 10 min
 * 7) Heat shock for 2 min at 42
 * 8) Put back on ice for 1 min
 * 9) For ampicillin selection, you can plate immediately, otherwise:
 * 10) Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 * 11) Plate on selective antibiotics, let incubate overnight

traA
I ligated my cleanup digestion: 6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase
 * Pound upside down on the bench to mix
 * Give it a quick spin to send it back to the bottom of the tube
 * Incubate on the benchtop for 30min
 * Put on ice and proceed to the transformation

I did a heat-shock transformation and plated on CA plates:
 * 1) Thaw a 200 uL aliquot of cells on ice
 * 2) Add 50 uL of water
 * 3) Add 30 uL of KCM salts
 * 4) Put your ligation mixture on ice, let it cool a minute or two
 * 5) Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
 * 6) Let sit on ice for 10 min
 * 7) Heat shock for 2 min at 42
 * 8) Put back on ice for 1 min
 * 9) For ampicillin selection, you can plate immediately, otherwise:
 * 10) Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 * 11) Plate on selective antibiotics, let incubate overnight

KILLR
I miniprepped the liquid culture that had been created from two of the colonies on my KA plate: MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
 * 1) Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
 * 2) Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
 * 3) Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
 * 4) Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
 * 5) Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
 * 6) Spin in centrifuge at top speed for 5 minutes.
 * 7) Label blue columns with an alcohol-resistant lab pen.
 * 8) Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
 * 9) Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
 * 10) Wash each column with 500 uL of PB buffer.
 * 11) Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
 * 12) Wash with 750uL of PE buffer (washes the salts off the resins).
 * 13) Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
 * 14) Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
 * 15) Label new tubes and put columns in them.
 * 16) Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
 * 17) Spin in centrifuge at top speed for 30 seconds.
 * 18) Take out columns and cap the tubes.
 * 19) Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

traA
I did not get any colonies from my previous transformation. I did another ligation using twice as much digested PCR product: 6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase I then transformed my bacteria: I set up another pcr reaction like I did on february 20th. 29 uL water 5 uL Expand 10x Buffer 2 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) 5 uL Oligo Oph22F (100uM) 5 uL Oligo Oph23R (100uM) 0.75 uL Expand Polymerase 1
 * Pound upside down on the bench to mix
 * Give it a quick spin to send it back to the bottom of the tube
 * Incubate on the benchtop for 30min
 * Put on ice and proceed to the transformation
 * 1) Thaw a 200 uL aliquot of cells on ice
 * 2) Add 50 uL of water
 * 3) Add 30 uL of KCM salts
 * 4) Put your ligation mixture on ice, let it cool a minute or two
 * 5) Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
 * 6) Let sit on ice for 10 min
 * 7) Heat shock for 2 min at 42
 * 8) Put back on ice for 1 min
 * 9) For ampicillin selection, you can plate immediately, otherwise:
 * 10) Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 * 11) Plate on selective antibiotics, let incubate overnight

KILLR
I digested my miniprep product so that I could run a mapping analytic gel: 6.5 uL ddH2O 2uL Miniprepped plasmid 1uL 10x NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)


 * Incubate at 37 on the thermocycler for 30 minutes
 * Run an analytical gel
 * Take a picture of the gel
 * Calculate the expected fragment sizes

My analytic gel again shows two different bands although both should be of the same length:

traA
I once again did not get any colonies from my tranformation. I begin to start over using the pcr product I made and cleaned up last class. I digest this product: 8uL of eluted PCR product 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI And Incubated for 1 hour at 37 degrees celsius I attempt to gel purify following this protocol:
 * All spins until the drying step are 15 second full speed spins.

However no band is seen for my product when I attempt to visualize my gel under the UV viewer.
 * 1) cut out bands minimizing extra gel matter.
 * 2) put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
 * 3) heat at 55, shake and/or vortex until the gel has dissolved.
 * 4) If the DNA is <300bp add 250uL of isopropanol
 * 5) transfer into the Zymo column inside a collection tube (small clear guys)
 * 6) spin through, discard waste.
 * 7) Add 200 uL of PE buffer (which is basically 70% ethanol)
 * 8) spin through, discard waste.
 * 9) Add 200 uL of PE buffer
 * 10) spin through, discard waste.
 * 11) spin for 90 seconds, full speed to dry.
 * 12) elute with 8.5 uL of water into a fresh Eppendorf tube

KILR
I set up another digest map of my miniprep DNA as the one I ran last class looked inconclusive. My product is in lanes 4 and 5 directly to the right of the ladder in lane 3. It is very hard to interpret this gel.

traA
I set up another digestion as my last attempt to gel purify failed. 8uL of eluted PCR product 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI And Incubated for 1 hour at 37 degrees celsius I then preformed a zymo cleanup and eluted my product:
 * 1) Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
 * 2) Transfer into the Zymo column (small clear guys)
 * 3) Add 500uL of Ethanol and pipette up and down to mix
 * 4) spin through, discard waste.
 * 5) Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
 * 6) spin through, discard waste.
 * 7) Add 200 uL of PE or Zymo Wash buffer
 * 8) spin through, discard waste.
 * 9) spin for 90 seconds, full speed to dry.
 * 10) elute with water into a fresh Eppendorf tube

KILR
I ran an analytic digestion of my miniprepped product: 6.5 uL ddH2O 2uL Miniprepped plasmid 1uL 10x NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)


 * Incubate at 37 on the thermocycler for 30 minutes
 * Run an analytical gel
 * Take a picture of the gel
 * Calculate the expected fragment sizes

A 1kb band a 2kb band are seen on the digestion which corresponds to my expected band sizes. I submit my part for sequencing. No picture is available as I used the UV lamp plate to visualize the bands.

traA
I ligated my cleanup digestion: 6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase
 * Pound upside down on the bench to mix
 * Give it a quick spin to send it back to the bottom of the tube
 * Incubate on the benchtop for 30min
 * Put on ice and proceed to the transformation

I did a heat-shock transformation and plated on CA plates:
 * 1) Thaw a 200 uL aliquot of cells on ice
 * 2) Add 50 uL of water
 * 3) Add 30 uL of KCM salts
 * 4) Put your ligation mixture on ice, let it cool a minute or two
 * 5) Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
 * 6) Let sit on ice for 10 min
 * 7) Heat shock for 2 min at 42
 * 8) Put back on ice for 1 min
 * 9) For ampicillin selection, you can plate immediately, otherwise:
 * 10) Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 * 11) Plate on selective antibiotics, let incubate overnight

I used contaminated LB medium during the rescue step as I did not check to make sure it wasn't cloudy.

KILR
I recieved my sequence back for the KILR part. Both sets of flanking restriction sites as well as the part are completely visible. The read is perfect and the part should be functional.

traA
I picked two colonies from my CA plate. There is evidence of contamination, however, a portion of my plate contained very low colony density and my two colonies were picked from this location. I put the liquid cultures in the upstairs labs. The protocol for picking colonies was to transfer LB medium with the correct antibiotic resistance to a test tube, use the micropipetter to pick one colony and drop the tip into the medium, and than transfer the test tube to the shaker. Extremely sterile technique was used.

traA
Gabe has very nicely done my miniprepping for my over the weekend. I ran an analytic digest on the first colony and saw the correct number of bands. 6.5 uL ddH2O 2uL Miniprepped plasmid 1uL 10x NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)


 * Incubate at 37 on the thermocycler for 30 minutes
 * Run an analytical gel
 * Take a picture of the gel
 * Calculate the expected fragment sizes

I submitted my first colony for sequencing.

traA
I recieved back my sequences, the entire part is visible and the BamHI, XhoI restriction site pair is visible as well. However the EcoRI site is not visible with a point mutation in the restriction site. It is extremely unlikely that this is actually a mutation as the analytic digest showed that the product was cut with EcoRI at this stage.