SBB12Ntbk-John Seggman

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John Seggman 02:23, 15 February 2012 (EST)

This is my next entry.

John Seggman 14:33, 16 February 2012 (EST)

Made 100uM stocks of oligos obb1226F and obb1226R

Prepared the following wobble reaction for SBB1226:

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

Made 100uM stocks of oligos araR-F and araR-R For oligos araR-F and araR-R, made an oligo dilution of:

9uL Water
1uL 100uM oligo

Prepared the following PCR reaction for SBB1201:

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

Discarded the remainder of the diluted oligos araR-F and araR-R per the "Cloning by PCR" protocol.

Followed all protocols exactly.

John Seggman 16:58, 17 February 2012 (EST)

Performed the Small-Frag Zymo Cleanup on SBB1226:

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 (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

Stored small-frag zymo cleanup product in the freezer for next time.

Next, loaded 6uL of SBB1201 PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel. Unfortunately, I am going to have to run the gel next time (not enough time today) so I will have to redo the loading of SBB1201 PCR product.

John Seggman 14:05, 21 February 2012 (EST)

Ran an analytical gel for SBB1201: Loaded 6uL of SBB1201 PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel. My band appears to match where I would expect my product to be on the gel. Therefore, my PCR worked.

Performed a wobble digest of SBB1226:

Set up the following reaction in a PCR tube:
50uL eluted DNA
5.7uL NEB Buffer 2
0.5uL NheI
0.5uL 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
  • Stored the post-digest 1226 product in freezer for next time

Performed a regular zymo clean-up on SBB1201:

  1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, discard waste.
  4. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  5. spin through, discard waste.
  6. Add 200 uL of Zymo Wash Buffer
  7. spin through, discard waste.
  8. spin for 90 seconds, full speed to dry.
  9. elute with 33uL water into a fresh Eppendorf tube

Stored post zymo clean-up in freeze for next time

John Seggman 14:22, 23 February 2012 (EST)

Performed a ligation reaction for SBB1226: Set up the following reaction:

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL NheI/BamHI 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

Performed a transformation for SBB1226 ~10 minutes after ligation reaction:

 1. Thaw a 200 uL aliquot of cells on ice
 2. Add 50 uL of water to the cells (if greater volume is desired)
 3. Add 30 uL of KCM to the cells 
 4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
 5. Add 70 uL of the cell cocktail to the ligation, stir to mix
 6. Let sit on ice for 10 min
 7. Heat shock for 90 seconds at 42 (longer incubation may work better)
 8. Put back on ice for 1 min
 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

Performed a NheI/BamHI digest of SBB1201:

Set up the following reaction:
8uL of eluted PCR product
1uL of NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI
  • Incubate at 37 degrees on the thermocycler for 1hr
  • Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees

Performed a zymo gel purification on SBB1201:

  • All spins until the drying step are 15 second full speed spins.
  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 (did not do this step)
  5. transfer into the Zymo column inside a collection tube (small clear guys)
  6. spin through, discard waste.
  7. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  8. spin through, discard waste.
  9. Add 200 uL of Zymo Wash Buffer
  10. spin through, discard waste.
  11. spin for 90 seconds, full speed to dry.
  12. elute with water into a fresh Eppendorf tube

Stored the zymo gel purification product for next time

John Seggman 19:01, 24 February 2012 (EST)

Picked 4 colonies from SBB1226 transformation:

  • 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 pipette tip
  • Drop it in the test tube
  • Incubate at 37 overnight

Performed a ligation reaction for SBB1201:

Set up the following reaction:
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL NheI/BamHI 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

Performed a transformation for SBB1226 ~5 minutes after ligation reaction:

 1. Thaw a 200 uL aliquot of cells on ice
 2. Add 50 uL of water to the cells (if greater volume is desired)
 3. Add 30 uL of KCM to the cells 
 4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
 5. Add 70 uL of the cell cocktail to the ligation, stir to mix
 6. Let sit on ice for 10 min
 7. Heat shock for 90 seconds at 42 (longer incubation may work better)
 8. Put back on ice for 1 min
 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

John Seggman 15:03, 28 February 2012 (EST)

Zach followed the "Picking of Colonies" protocol for part SBB1201. Both products, SBB1201 and SBB1226, showed no growth. My plate for SBB1226 had no red colonies. It is possible that I used the wrong vector. My plate for SBB1201 had large red colonies but small white colonies. All 4 colonies that were picked for SBB1201 were very small white colonies potentially indicating contamination rather than my bacteria of interest. Because both products did not grow in the test tubes, I discarded them and re-did the ligation and transformation reactions for both products today, making absolutely sure to use the PBca9525 NheI/BamHI vector.

Ligation protocol:

Set up the following reaction:
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL NheI/BamHI 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 protocol:

 1. Thaw a 200 uL aliquot of cells on ice
 2. Add 50 uL of water to the cells (if greater volume is desired)
 3. Add 30 uL of KCM to the cells 
 4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
 5. Add 70 uL of the cell cocktail to the ligation, stir to mix
 6. Let sit on ice for 10 min
 7. Heat shock for 90 seconds at 42 (longer incubation may work better)
 8. Put back on ice for 1 min
 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

John Seggman 23:54, 1 March 2012 (EST)

Zach picked the colonies for both products, SBB1201 and SBB1226 (2 colonies for each product). All 4 colonies grew in the test tubes.

Proceeded to do a mini-prep for all 4 samples (1201-1, 1201-2, 1226-1, and 1226-2):

  1. Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube.
  2. Dump supernatant
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
  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 full speed for 15 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 full speed for 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 full speed for 15 seconds and flick out liquid again.
  14. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
  15. Label new Microcentrifuge tubes and put columns in them.
  16. Elute them by adding 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.

After completing the mini-prep purification for all 4 samples, there wasn't enough time to run an analytical digest. Used the remaining time to decide that for SBB1201, will be using a NheI/BamHI digest and for SBB1201, a BgIII/BamHI digest.

For SBB1226, the reason I chose BgIII (as opposed to NheI) is because if NheI was used, the digest product would be too small to be detected on a gel

John Seggman 16:10, 2 March 2012 (EST)

Started the Analytical Digest (Mapping) protocol for all 4 samples (1201-1, 1201-2, 1226-1, and 1226-2):

2x Set up the following 10uL reaction in a PCR tube:

6uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
.5uL BgIII (for 1226)
.5uL BamHI 

Incubate at 37 on the thermocycler for 30 minutes

2x Set up the following 10uL reaction in a PCR tube:

6uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
.5uL NheI (for 1201)
.5uL BamHI 

Incubate at 37 on the thermocycler for 30 minutes

Did not have time to run a gel so prepared the 4 mini-prepped products to be loaded onto a gel for next time (mixed 4uL loading buffer and 6uL mini-prepped product in a PCR tube).
Stored samples in freezer for next time.

John Seggman 15:04, 6 March 2012 (EST)

Finished the Analytical Digest (Mapping) protocol for all 4 samples (1201-1, 1201-2, 1226-1, and 1226-2):

  1. Loaded my mini-prep products (1201-1, 1201-2, 1226-1, and 1226-2) onto a gel.
  2. Somebody took a picture of the gel.
  3. Calculated the expected fragment sizes: 1201- 1092 + 627; 1226- 1048 + 1640
  4. Are the calculated sizes consistent with the bands on the gel?
  • NO for 1226-2. For 1201-1 and 1201-2, they may or may not be the actual part, sbb1201. 1226-1 did not show up on the gel.

Image:NEB_2-log_ladder.gifImage:2012_03_06_gel2_ssb2012spring.jpg

Lanes:

1 2 3 4 5 6 7 8 9 10
AJ A+BJS 1201-1JS 1201-2JS 1226-1JS 1226-2LadderPR 02-1PR 02-2PR 30-1JK 1233-1

Going to redo the digests on Thursday.

John Seggman 18:22, 8 March 2012 (EST)

Redid the Analytical Digest protocol for all 4 samples again to make sure that I did not do something wrong and/or to see if stopping midway through the protocol had an adverse effect on the gel.

Image:NEB_2-log_ladder.gifImage:2012_03_08_gel2_ssb2012spring.jpg

  • Obtained the same results for the 1226 samples: For some reason, 1226-1 did not show up on either gel and 1226-2 is not the expected sizes, 1048 +1640.
  • For 1201-1 and 1202-2, the results appear to be similar to the first gel. After checking with fellow classmates what they expected their band sizes to be on that first gel, March 8 Gel 2,the professor and I determined that what we were seeing was sbb1201.
  • Submitted 1201-1 and 1201-2 for sequencing

John Seggman 13 March 2012 (EST)

  • Unfortunately, was out sick for this day.

John Seggman 15 March 2012 (EST)

  • Decided to redo the ligation and transformation protocols for sbb1226 in an attempt to see if this is why sbb1226 was not appearing to work based off the results of the analytical digest:

Ligation protocol:

Set up the following reaction:
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL NheI/BamHI 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 protocol:

 1. Thaw a 200 uL aliquot of cells on ice
 2. Add 50 uL of water to the cells (if greater volume is desired)
 3. Add 30 uL of KCM to the cells 
 4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
 5. Add 70 uL of the cell cocktail to the ligation, stir to mix
 6. Let sit on ice for 10 min
 7. Heat shock for 90 seconds at 42 (longer incubation may work better)
 8. Put back on ice for 1 min
 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

John Seggman 20 March 2012 (EST)

  • Zach picked 4 colonies of sbb1226 the day before; one colony grew in solution
  • Spun down the cells and discovered that the cells were pink
  • This was the 2nd attempt to religate and since it failed to work (cells were supposed to be white), I joined sequencing team B because it would be too late to start over
  • Finished sequencing

John Seggman 22 March 2012 (EST)

Came into lab but since our group was done with sequencing, there was nothing to do so I left

John Seggman 3 April 2012 (EST)

lecture + team 5 planning; started working on the protocol

John Seggman 5 April 2012 (EST)

Polished up the protocol:

  • Note- Au and I were the only ones in our group who researched and developed the protocol because other 2 members joined late.

Detailed Protocol: 1. Transform experimental and control plasmids into E coli (with the ctx promoter driving GFP expression, this e coli does not have spec, has amp and cm, ), plate on relevant antibiotics (spec+ either amp or cm) 1. use transformation protocol online 2. plasmids: 1. experimental (FKBP, pBca9525-sbb1224) 2. no toxR, yes specR 3. yes toxR (functional) 4. yes toxR (not functional) 3. reporter plasmid (Pctx-sfGFP) is pBJH1601CA-BssS2 2. pick good colonies and grow in liquid culture overnight (with spec) 1. use transformation protocol online at http://openwetware.org/wiki/Template:SBB-Protocols_Micro1 2. make sure your colonies. Looks normal compared to others, (not green, size, #) 3. next day, dilute ~1:100, put in 96 well plate, and grow in different concentrations of B/B+spec&amp/cm 1. dilute using new media+B/B+spec&amp/cm (calculate how concentrated B/B needs to be) 2. the awesome machine will incubate and take measurements at the right time as long as you program it right. 3. Manual from B/B producer suggests 30min to 12+hours 4. 3 hours if probably enough (professor says), very little is slower than 20minutes in E coli, GFP maturation takes ~40min) ← nvmd, we'll be doing it for 12 hrs 5. ***also do growth curve measurements during this time! (at different time points) 1. half hour increments for 12 hrs total 2. actually, let the machine run overnight (schedule it!); cells need time to get out of stationary phase

John Seggman 10 April 2012 (EST)

Transformed the cells with the five different plasmids
Plasmids used:

  • reporter plasmid: Bsrs52
  • experimental plasmid (FKBP): pBca9525-sbb1224
  • Control- non toxR, just specr (spec): ZNR104a
  • Control- always on functional toxR(FtoxR):Bgc0005
  • Control- always off nonfunctional toxR (NFtoxR): 9525-1846

1) Transformation Protocol for transformation

  1. Thaw a 150 uL aliquot of cells (with reporter plasmid not already in them) on ice
  2. Do Not Add 50 uL of water to the cells (if greater volume is desired)
  3. Add 30 uL of KCM to the cells 
  4. Put your diluted plasmids on ice, let cool a minute or two (plasmids already diluted)
  5. Add 70 uL of the cell cocktail to the 1 or 2uL of each plasmid (reporter + exp or control), stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour

2) Make B/B solution to spread onto plates

 add .5uL of stock B/B into 2.2mL
 aliquot .5mL onto each plate, and spread by tilting (not using the metal spreader)

3) Spread 70uL of each tube (8 total) onto B/B and no B/B plates

John Seggman 12 April 2012 (EST)

Redid the transformation because we didn't actually transform with reporter plasmid in first transformation.
Plasmids used:

  • reporter plasmid: Bsrs52 (this time, add 2uL of this instead of 1uL)
  • experimental plasmid (FKBP): pBca9525-sbb1224
  • Control- non toxR, just specr (spec): ZNR104a
  • Control- always on functional toxR(FtoxR):Bgc0005
  • Control- always off nonfunctional toxR (NFtoxR): 9525-1846

1) Transformation Protocol for transformation

  1. Thaw a 150 uL aliquot of cells (with reporter plasmid not already in them) on ice
  2. Do Not Add 50 uL of water to the cells (if greater volume is desired)
  3. Add 30 uL of KCM to the cells 
  4. Put your diluted plasmids on ice, let cool a minute or two (plasmids already diluted)
  5. Add 70 uL of the cell cocktail to the 1 or 2uL of each plasmid (reporter + exp or control), stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour

2) Make B/B solution to spread onto plates

 add .5uL of stock B/B into 2.2mL
 aliquot .5mL onto each plate, and spread by tilting (not using the metal spreader)

3) Spread 70uL of each tube (8 total) onto B/B and no B/B plates

John Seggman 13 April 2012 (EST)

Zach and I picked the colonies. I jotted down the results:

NFtoxR w B/B NFtoxR wo B/B FtoxR w B/B FtoxR wo B/B FKBP w B/B FKBP wo B/B specR w B/B specR wo B/B
whitewhiteno colonesgreengreengreenwhitewhite


Analysis of Results:
From Au's notebook because I couldn't have said it better: All exhibited correct phenotype except for FKBP (experimental plasmid; should have been white without B/B, but were green on both B/B and non B/B plates) and FtoxR (no colonies on B/B plate and ~6 colonies on non B/B plate) - it's possible that the FKBP results were due to cross contamination on the plasmid plate (there was condensation/liquid on the top of the wells/cover) so we will retransform

John Seggman 17 April 2012 (EST)

  • re-transformed B3 on B/B because colonies were green. they were supposed to be white
  • .25 ul of BB in 1.1 ml of water
  • also retransformed function ToxR because only 4 colonies showed up

John Seggman 24 April 2012 (EST)

  • organized TECAN data so we could graph it
  • decided to work on presentation via email as opposed to coming in on Thursday
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