SBB09Ntbk-Roger Lowe

EeaA Intimin gene part construction

February 18, 2009 Created cloning solution for EaeA Intimin gene using buffer, primers, expand polymerase, and template DNA.

PCR reaction

*Make 100uM stocks of primers, this is the concentration used directly for the reaction
*Prepare the following reaction:
  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 1 (100uM)
  5 uL Oligo 2 (100uM)
  0.75 uL Expand Polymerase 1

Ran on gel to check size/ presence of EaeA gene. The gene ran slightly further than expected, suggesting that it could be smaller than the hoped for gene.

February 25, 2009

Regular Zymo Cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

#Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 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

During this purification I put my reaction tube in the centrifuge unlabelled and it got confused with Nihkeil. We reran our samples side by side on a gel, and found which sample was my EaeA part.

Electrophoresis

2ul EaeA PCR sample
1ul Dye

February 27, 2009 In our gel results, my protein ran to about 1.7 kb, Nikhil's did not show, so we concluded that since my gel ran to about the expected length of my gene, I had grabbed my gene, EaeA intimin. I continued on to ligating my gene to the pBca9495CA plasmid.

Ligation of EcoRI/BamHI digests

*Set up the following reaction:
  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

Transformation by heat-shock

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

#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

Picking of colonies

    * For each construct you will pick and later miniprep 2 colonies
    * Add 4mL of LB media with the appropriate antibiotics to a clean test tube
    * Pick a well-isolated, round, and "normal" looking colony with a toothpick
    * Drop it in the test tube
    * Incubate at 37 overnight 

Miniprep purification of DNA

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 I did not get any colonies for EaeA-CA transformed plasmid. I started again at ligation with the digested and cleaned EaeA plasmid. My ligation and transformation reactions followed the same procedures as the ligation and transformation protocol listed above.

I also started a new EaeA intimin PCR reaction in case my second plating efforts do not produce any colonies, using the same PCR protocol as explained above.

Aluminum Binding Protein gene part construction

February 18, 2009 I started my wobble reaction with the oligos designed and ordered the week before.

Wobble Reaction

The Wobble procedure is a variation of Klenow Extension that begins with two oligonucleotides that overlap by around 20 bp on their 3' ends and uses a thermostable polymerase

• Order the oligos, they don't need to be purified in any special way, smallest scale is ok
• Make 100uM stocks, this is the concentration used directly for the reaction
• Prepare the following reaction:

  
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 1 (100uM)
  5 uL Oligo 2 (100uM)
  0.75 uL Expand Polymerase 1

• Run the wobble program, whick is:

 2 min at 94
 10 cycles of:
 30 sec at 55
 30 sec at 72
 (or something similar)

• There is no point in running an analytical gel afterwards, there is nothing to see
• You'll want to run short fragment cleanups to remove the polymerase prior to digestion steps

February 23, 2009 After performing Klenow extension for aluminum binding gene, I performed Zymo Cleanup.

Small-Frag Zymo Cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

#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

Now to be cyclized

Created solution for Klenow (wobble) extension for aluminum binding protein, now being cyclized.

I then continued to digest the aluminum binding peptide gene.

EcoRI/BamHI Digest of Wobble Products

For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.

 50uL eluted DNA
 5.7uL NEB Buffer 2
 1uL EcoRI
 1uL BamHI

   * Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
   * Incubate the reaction at 37 degrees on the thermocycler
   * Proceed to another Zymo small fragment cleanup

February 25, 2009

Ligation of EcoRI/BamHI digests

• Set up the following reaction:

  
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

Transformation by heat-shock

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

#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

Picking of colonies

• For each construct you will pick and later miniprep 2 colonies
• Add 4mL of LB media with the appropriate antibiotics to a clean test tube
• Pick a well-isolated, round, and "normal" looking colony with a toothpick
• Drop it in the test tube
• Incubate at 37 overnight

February 27, 2009

My two colonies for ABP in 9495-CA plasmid were labeled bomb 1 and bomb 2. I mini-prepped both of them.

Miniprep purification of DNA

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 02, 2009 I digested my isolated plasmids using EcoR1 and BamH1.

Plasmid Digestion

5ul dH20
3ul DNA solution
1ul NEB buffer
0.5 ul EcoR1
0.5 ul BamH1

Both of my samples showed plasmid backbones that ran on the gel, which indicates that the plasmids did indeed transform into the cells.