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

<pre>
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

February 23 , 2009

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:

#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#elute with water into a fresh Eppendorf tube

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.

KILLR

I cleaned up my pcr product:

#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.

For my wobble products, I digested the entire extension reaction-worth of DNA.

*I Set up the following reaction in a PCR tube:
  50uL eluted DNA
  5.7uL NEB Buffer 2
  1uL EcoRI
  1uL BamHI
*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:

#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.

February 25, 2009

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:

#Add 180 uL of Zymo ADB buffer (brown bottle) to a 50uL reaction.
#Transfer into the Zymo column (small clear guys)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#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:

#Thaw a 200 uL aliquot of cells on ice
#Add 50 uL of water
#Add 30 uL of KCM salts
#Put your ligation mixture on ice, let it cool a minute or two
#Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
#Let sit on ice for 10 min
#Heat shock for 2 min at 42
#Put back on ice for 1 min
#For ampicillin selection, you can plate immediately, otherwise:
#Add 100uL of LB, let shake in the 37 degree incubator for 40 min
#Plate on selective antibiotics, let incubate overnight

February 27, 2009

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:

#Thaw a 200 uL aliquot of cells on ice
#Add 50 uL of water
#Add 30 uL of KCM salts
#Put your ligation mixture on ice, let it cool a minute or two
#Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
#Let sit on ice for 10 min
#Heat shock for 2 min at 42
#Put back on ice for 1 min
#For ampicillin selection, you can plate immediately, otherwise:
#Add 100uL of LB, let shake in the 37 degree incubator for 40 min
#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)''
#Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
#Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
#Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
#Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
#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.
#Spin in centrifuge at top speed for 5 minutes.
#Label blue columns with an alcohol-resistant lab pen.
#Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
#Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
#Wash each column with 500 uL of PB buffer.
#Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
#Wash with 750uL of PE buffer (washes the salts off the resins).
#Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
#Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
#Label new tubes and put columns in them.
#Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
#Spin in centrifuge at top speed for 30 seconds.
#Take out columns and cap the tubes.
#Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

March 2, 2009

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
*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 then transformed my bacteria:

#Thaw a 200 uL aliquot of cells on ice
#Add 50 uL of water
#Add 30 uL of KCM salts
#Put your ligation mixture on ice, let it cool a minute or two
#Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
#Let sit on ice for 10 min
#Heat shock for 2 min at 42
#Put back on ice for 1 min
#For ampicillin selection, you can plate immediately, otherwise:
#Add 100uL of LB, let shake in the 37 degree incubator for 40 min
#Plate on selective antibiotics, let incubate overnight

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

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:

March 4, 2009

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.

#cut out bands minimizing extra gel matter.
#put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
#heat at 55, shake and/or vortex until the gel has dissolved.
#'''If the DNA is <300bp''' add 250uL of isopropanol
#transfer into the Zymo column inside a collection tube (small clear guys)
#spin through, discard waste.
#Add 200 uL of PE buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#elute with 8.5 uL of water into a fresh Eppendorf tube

However no band is seen for my product when I attempt to visualize my gel under the UV viewer.

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.

March 6, 2009

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:

#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#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