Team:Michigan/Pili August September

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8/7/2010

Kevin, Marc, Alena

PCR #1 Used a gradient from 40ºC to 60ºC for the first 3 cycles to find the optimum annealing temperature.

All of the annealing temperatures gave a good result according to the gel.

8/7/10 notes

8/9/2010

Kevin, Marc, Alena

Used a 57ºC degree annealing temperature to get enough DNA for the digest and ligation.

4 out of the 5 PCR reactions worked well according to the gel.

The 5th well could have been a loading problem or there wasn't enough DNA.

8/9/10 notes

8/11/2010

Kevin, Marc

Met with Chris, received advice for updating digest and ligation protocols.

8/12/2010

Kevin, Marc, Alena

Met and discussed protocols for digestion and ligation of FIMB into pBAD.

1. Added 5 mL of LB broth each to 2 50 mL falcon tubes in the ERB lab using sterile technique.

2. Added 5 microliters of Kanamycin to each of the 50 mL tubes in step 1.

Went to the budget committee meeting for 1 hour with the tubes.

3. Obtained the cryostock of pBAD from the Lin -80C freezer (iGEM box cell #73)

4. Stabbed cryostock using a sterile 200 microliter pipette tip and pipetted into media from step 2.

5. At 8:05PM placed the two falcon tubes from step 4 into the incubator/shaker at 30ºC.

8/14/2010

Kevin, Marc, Alena

Miniprep pBAD plasmid

1. inoculate 5.0mL LB in 50mL conical tubes w/ 100ug/mL of ampilicin
   1. the cultures (2 of them) grew for ~12hrs (8am to 8pm)
2. centrifuge (using the 50mL conical tubes) for 5000rpm for 10min at 4ºC
3. carefully discard the supernatant and resuspend the pellet with 250uL of P1 buffer (kept in the 4ºC fridge)
4. transfer into a labelled 1.5mL eppendorf tube (set pipetman to 500uL just in case)
5. add 250uL P2 Buffer--> invert 4-6times (should turn blue)--> add 350uL N3 buffer--> invert 4-6times (should become vicious or clumpy)
   1. centrifuge at 13,000 rpm for 10 min
6. pipette out supernatant into a QIA spin column--> centrifuge for 60s--> discard flow through
   1. did not pipet out all of the supernatant
7. add 500uL PB buffer --> centrifuge 60s--> discard flow through
8. add 750uL PE buffer--> centrifuge 60s--> discard--> centrifuge 60s again
9. transfer into a labeled 1.5mL eppendorf tube--> add 50uL EB (elution buffer)
   1. allow it to sit for 1 min (it helps to release the DNA from the column)

fimB PCR product Purification

1. Used sample A and B of PCR product (save C and D for later)
   1. total volume of PCR product = 81 uL (40.5uL separately)
2. add 5 volumes of PB buffer (202.5uL) to 1 volume of PCR product (40.5uL); invert
3. transfer into a QIAquick spin column (provided in the Qiagen kit)
   1. set pipetman to 260uL to be sure to get all of the mixture
4. centrifuge spin column at 13,000rpm for 60s
5. discard the flow through (in the collection tube) and add 750uL PE buffer (to wash the DNA)

repeat step 4 again

1. discard flow through and centrifuge again to get the remain buffers out
2. place the column into a labeled 1.5mL eppendorf tube
3. add 50uL EB buffer (to elute out the DNA) directly to the white inner circle of the column (avoid touching the pipet tip to the column)
   1. allow the mix to sit in the column for 1 min, then centrifuge for 1 min (13,000rpm)
   2. remember to point the cap of the eppendorf tube in the opposite direction of the centrifuge machine

fimB Digest

1. pre-programmed PCR machine to Digest (DIG1)
2. use 5 PCR reaction tubes for each; 5 for fimB and 5 for pBAD
3. add the following amounts in that order (total volume of 20uL)

• 16 uL of the DNA (fimB and pBAD to their respective tubes)
• 2 uL of NEB 2 buffer
• 1 uL NcoI
• 1uL HindIII

1. incubate for 37ºC overnight (12hrs)
   1. place into 4ºC fridge the next day

8/16/2010

Kevin, Marc, Alena

Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store

Attempted experiment:

• Added 1uL of CIP (Calf Intestine Phosphatase) to plasmid reactions only (pBAD)
  • CIP will cut off the 3' phosphate to prevent the plasmid from folding back on itself
• incubate CIP-pBAD at 37C for 1 hr and then heat-shock (65ºC for 15 min)

We made a huge human error when setting up our thermal cycle program on the PCR machine. We did not realize we only set the thermal cycle for our digest on 8/14/2010>> [1] for 12 minutes (12:00) rather than the intended time period of 12 hours (12:00:00). This explains the condensation present in the PCR machine (it stayed at 4C for too long). We discovered our mistake after running the incubation of CIP+pBAD for 37C for (1:00) 1 minute then heat-shock at 65C for 15 minute. It is VERY IMPORTANT to triple check one another when entering in any program.

Rest of the day:

• headed over to the ERB and made two cultures of 5mL LB-Amp (in 50mL conical tubes)--> placed in the Lin Lab 4ºC fridge

8/17/2010

Kevin, Marc, Alena

Marc inoculated the two cultures made last night (8/16/2010) with pBAD and grew in 37C shaker (~9am to ~6p, 9hrs)

NOTE REGARDING LB CULTURE MADE ON 8/16/2010

• Marc noticed the 400mL LB media made in the ERB to be contaminated.
• Our two cultures could have been contaminated but pBAD grew pretty well (saturated) so we're not too worried

fimE and fimB knock out (K.O.) strains came in

• Marc adds 50uL of kanamycin onto each LB-agar plate and spread evenly with sterile glass beads
  • LB-agar plate ~25mL
  • Jeremy Minty's advice: more Kan is better than too little --> Kan 100 instead of Kan 50
  • allow the plate to absorb the kanamycin for 2 hours before applying the strains
• Alena follows Jeremy Minty's example of carefully taking out the small filter circle out of the foil with sterile techniques onto a LB-agar plate (previously labeled)
  • add ~75uL of LB onto the filter circle
  • streak the filter paper w/ inoculating streakers (used 3 of them)
• incubate plates w/ filter paper (still on the agar) upside (agar side up) at 37C overnight

Followed 8/14/2010 protocol (above) for:

• Miniprep for pBAD plasmid
  • Last time we used the incorrect spin column (QIAquick spin column; purple; used for PCR purification). This time we used QIAprep spin column (blue; had no cap on the column)
• PCR purification for fimB
  • used PCR products labeled C and D tubes
• Digest fimB and pBAD
  • This time, made sure the time on the program was set to 12:00:00
  • 5 rxns for each fimB and pBAD (10 total tubes, 20uL total volume in each tube)

8/18/2010

Kevin, Marc, Alena

Met with Chris to discuss about yeast agglutination

• Chris sent us a possible paper with a decent protocol to look at for the assay Media:YeastAgglutinationPaper.pdf

NOTE ABOUT K.O. STRAINS

• fimE K.O. did not grow out (which contradicts the prediction that fimE K.O grows faster than fimB K.O.)
• try letting the plate grow at 37C for a longer period of time (one colony observed)
• remove the filter circle paper and put onto a new LB-agar plate
• suspect too much kanamycin on the plate

Lab work:

1. add CIP (calf intestine phosphatase= cuts off the 3' phosphate to prevent plasmid from closing back on itself) to plasmid (pBAD) reaction tubes only--> incubate at 37C for 1 hour (using PCR machine)
2. heat/inactivate pBAD and fimB reaction tubes for 15 min at 65C (again using PCR machine)
3. perform DNA purification on the digests (follow 8/16/2010 procedure)
   1. using 210uL and 200uL of PB buffer for pBAD and fimB respectively
4. ran a gel on the digest

1. ran nanodrop3.0.1 on the digest
   1. use EB buffer as the blank
   2. ALWAYS clean twice when done using the machine

8/22/2010

Kevin, Marc

Ran Ligation of FimB and pBAD for 16 hrs O/N

Prepared culture of DH5a for electroporation tomorrow.

8/23/2010

Kevin, Marc

Growing competent cells for electroporation.

Colony started growing at 2:00.

Measured ODs

Precipitate Ligation product w/butanol

Add to eppendorf tube:

1. 50 ul ultrapure H2O
2. Ligation product
3. 500 ul butanol

Spin at 4C, 13000 rpm for 20 min

Made ampicillin plates

• Add 25 ul Amp100 to LB plates

6 plates:

• Cells only
• Plasmid only
• 2x pBAD+FimB undiluted
• 2x pBAD+FimB 1:100

9/2/2010

Kevin

Electroporation of pBAD+FimB into K12 and ΔFimB::kan cells.

15 plates
1x K12 control (amp)
1x Plasmid control (amp + kan)
1x ΔFimB control (amp + kan)
6x ΔFimB (amp + kan)
6x K12 (amp)

9/7/2010

Kevin

Started overnight cultures of 1:1000 dilutions of K12 and ΔFim::kan, both with the pBAD+FimB plasmid.

9/8/2010

Kevin

Created frozen stocks of K12 and ΔFim::kan w/the plasmid from overnight cultures. Stored them in box 1 in the ERB -20C freezer.

Ran miniprep of K12 and ΔFim::kan, using 5 ml of each culture. Note: Centrifuge in ERB will only go up to 5000 rpm w/50 ml tubes, therefore we transferred the culture to several eppendorf tubes. This was probably not a good idea, because we were left with a lot of leftover supernatant and that could have diluted the buffers. In the future, we should centrifuge the culture 1 ml at a time, and add 1 ml after each cycle.

Stored the product from miniprep in box 2 in the ERB -20C freezer.

9/9/2010

Kevin

Cryopreserved frozen stocks of K12 and ΔFimB::kan w/the plasmid.

Ran digest to determine whether the plasmid and FimB were actually in the cell. Used protocol from previous digest on 8/14.

9/10/2010

Kevin

Ran gel of previous day's digest, unfortunately the gel was inconclusive. There is an undetermined error with the gel electrophoresis machine.