My Notes II

From 2008.igem.org

Latest revision as of 07:56, 6 September 2008

24 August 2008 (Su)

• Ran ELISA for sca40-43. (only ran with 3 antibodies: AP, HA, and FLAG. -- out of myc antibody)
• IT WORKED. (partially)
• Results:
  • C = clear
  • L = light yellow
  • M = medium yellow

Conclusion:

• sca40 (AP tag) consistently bound with AP and HA antibody
• sca41 (HA tag) consistently bound with HA antibody
• sca42 (myc tag) was inconclusive, since there was no myc antibody as a positive control
• sca43 (FLAG tag) consistently bound with FLAG antibody; but also displayed binding with AP and HA antibodies

Next, hopefully someone will run another ELISA with more concentrated cultures, since the 10x diluted cultures did not turn yellow as expected. We assume that the washes might have lowered the concentration of either tags or culture. Hopefully, increasing the initial concentration will counteract the washes.

As for me, my last day at lab is complete. =) I am happy to have completed 2 ELISAs in time...

23 August 2008 (Sa)

I planned to come in to run a 2nd trial ELISA for sca40-43...but apparently, someone forgot to grow up cultures for me to test with the night before! I grew up cultures (added arabinose)and will use them in ELISA test tomorrow.

20 August 2008 (W)

• Came in at 9:30am -- Set up 3rd time transformation of sca49-sca52
• Jin finished plating later in the day

19 August 2008 (T)

• checked PCR, didn't turn out right -- no band
• reset up PCR of AKL41. Finished all the way up to ligation. Put ligations in freezer before I left; set up transformation tomorrow.
• Ran an ELISA for sca40-sca43 in MC1061 --- which ended at 11p.m. Results were not good...

Additionally, I'm feeling a little disoriented, considering that today is my last official day in lab... =(

18 August 2008 (M)

• didn't get to run an ELISA on sca40-43 (couldn't find all the antibodies)
• do another phoA assay for sca49-52, pick 2 yellow samples, miniprep, send to seq; and transform into MC1061
  • sca49-52 did not turn yellow
  • colony pcr with sca49 1-8, run on gel to check size; supposed to be ~3000bp, came out ~1500 (only righty/vector part)
• started to re-redo sca49-sca52, with newly digested righty/vector parts sca44-sca47; and redone PCR of AKL41

17 August 2008 (Su)

Wasn't in lab today.

• Jin picked 12 colonies each of sca49-52
• and picked MC1061 colonies of sca40-43

16 August 2008 (S)

• sca40-43 did NOT grow, because I accidently put them on the wrong double-antibiotic plate
• MC1061 tests were good
• phoA assay: sca49-sca52 did not turn yellow when the controls did
• redo sca49-sca52 from PCR product. zymoed, digested, zymoed, ligated with sca44-sca47, transformed, and plated on AK.

15 August 2008 (F)

• picked colonies for sca49-52 -- 12 each; I again got to use 2 blocks simultaneously
• also picked colony for positive controls AKL41 and AKL42
• checked on whether MC1061 cells grew in good media...(not contaminated) -- made competent cells.
• transformed sca40-sca43 into MC1061
• test for competence in parallel

14 August 2008 (Th)

• 1st thing in the morning: aliquot 5 ul of my basic parts and Cici's basic parts to send to the registry.
• got sequencing results for my mislabeled "yellow samples" sca42-5 and 43-2.
  • realized that I don't have a good yellow sample of myc! tag
  • took 3 remaining yellow samples for sca42 (colonies 7, 8 and 12)
  • also picked 3 corresponding colonies off the plate to grow in media, just in case my miniprep samples don't work.
• got sequencing results for sca44-47: there was a slight mix-up of tubes and tube numbers, but the samples themselves all turned out to be perfect.
  • Will resequence the mixed-up tubes/numbers for clarification.
  • meanwhile, still digested sca44-47 as "righty part + vector", zymoed; then ligated 1ul of sca44-47 with zymoed sca48 part from Tuesday = sca49, sca50, sca51, sca52; transformed
  • Jin plated sca49-52

13 August 2008 (W)

• got sequencing results for sca40-43
  • seems that I have one good final product for every tag
  • possible tube mislabeling: what was labeled sca42-5 came out to be what I expected in sca43(colony 2)

...will send sca42-5 and sca43-2 to seq again.

• Check plate for sca44-47 for co-transformation - I have at least 2 not cotransformed of each part (yessssssss!)
• Ran colony PCR for all 24 samples of sca44-sca47 - it's the first time I've put 24 tubes in the thermocycler!
• Ran a gel for all 24 samples - the longest gel I've ever run. The strange thing is that ALL 24 samples (even the ones that were "supposedly cotransformed" looked "right" on the gel.
• selected 2 colonies for sca44-sca47 each to miniprepp;sent for sequencing
• grew up fresh MC1061 to transform sca44-47 into

12 August 2008 (T)

• sca48 PCR did not work.
  • re-diluted 100uM pelB>R oligo
  • set up 2nd PCR
  • gel check = correct!
  • purified (by zymo), digested, and zymo 2nd time
• picked colonies for sca44-47, screen on ACK plate
• sca40-43 (complete final products):

1. did phoA assay to check for yellow signal
   1. three results: clear, light yellow, or REALLY yellow.
   2. all 4 different products each had at least 2 light/really yellow samples.

Miniprepped:

sca40-1  -- Really yellow  seq
sca40-4  -- light yellow   seq
sca41-4  -- really yellow  seq
sca41-8  -- Really yellow
sca42-2  -- Really yellow  seq
sca42-5  -- Really yellow  ?
sca43-2  -- Really yellow  ?
sca43-4  -- light yellow   seq

11 August 2008 (M)

• pick 12 colonies each for sca40-sca43. For the first time, I will simultaneously need two 24-well blocks...
• miniprep sca4-sca7 (no mapping or sequencing necessary)
• Assemble Layer 1 of Branch 2: {<tag>}.{<phoA!}.{b1006}

=sca44. sca45, sca46, sca47

• make sca48 {Pbad}.{rbs_pelB>} from AKL41: set up PCR with pelB> reverse oligo.

10 August 2008 (Su)

• Came into lab today!!!
• Moved sca4-sca7 into Lefty cells instead of righty. (already in AC vector, still useful)
• Redid everything from yesterday, only using AKL 41 as the "Aron part"
  • PCRed AKL 41 with ca998F and mea37R (phoA> reverse) oligos to make sca39: {pBad}.{rbs_pelB}.{phoA>}
  • assembled sca39 with {<tag!}.{b1006} parts sca22, sca23, sca24, sca25.
  • completed assemblies = sca40, sca41, sca42, and sca43.

09 August 2008 (S)

Busy…very busy…

• Miniprepped 4 colonies of fimH;
• Simultaneously did 3 different types of digestion protocols (with 10 tubes): 1.)mapping 2.)digesting vectors, and 3.)digesting Aron’s parts
• Digestion mapped all 4 miniprepped fimH's. All had different band sizes… sent colony 3 for sequencing.

• Finished up with 16 and 19, each with my parts sca22-25

= total 8 things.

• Left before transformation and plating, but found out in evening that 16 and 19 parts were unnecessary, since Aron had an even more complete part (with pBad promoter)… didn’t plate.

08 August 2008 (F)

• Sca2: both diluted colonies co transformed -- no miniprepping...
• FimH (SC43) came back bad read
• Took fimH from Jin’s Amp plate (same day as re-amplified fimH), picked 4 colonies and grew in LB Amp-Checked plate and culture for amplifying Bca9194 - nothing grew.
• Ran a gel in the morning at 9 a.m. -- gel turned out weird: 2 bands - one at 4000 and other at 650-750ish bp both were wrong… (size was supposed to be 1610 bp)
• talked with Chris and Jin. Stopped using questionable Bca9194; used Aron’s parts - AKL16, 17, and 19 - as PCR template instead.
• Ran gel to check Aron’s 16, 17, and 19 -- used PCR product of AKL16 and AKL19
• Did a zymo for AKL 16 and AKL 19.

07 August 2008 (Th)

• Picked colonies for sca2-7; streaked on CK plate, screened on Amp. Growing overnight in CK media
• mini meeting - new methods enforced due to assembly status:

Assembly Status:

All transfers completed successfully, EXCEPT sca2 (rbs1-A) and sca3 (fimH)

Layer one assemblies completed:
sca21-{<phoA!}.{b1006}
sca22-{<AP!}.{b1006}
sca23-{<HA!}.{b1006}
sca24-{<myc!}.{b1006}
sca25-{<FLAG!}.{b1006}

None of Layer 2 or 3.

• Decided to shrink old tree and use new methods to finish the tags: attach my {<tag!}.{b1006} parts to {FimH}.{phoA!}
  • Take existing {FimH}.{PhoA!} (aka. pBca1102-Bca9194), set up PCR using ca998 oligo and mea37 (<phoA> reverse oligo) to remove stop codon
  • At first, the PCR product came out mysteriously large. We tried 3 times and the same thing occurred.

It was not until 11:00pm, that figured that I was using the wrong oligo ... we set up the 4th PCR then - with the correct oligo.

• also amplified Bca9194 for personal stock, since we were running low - grew on amp plate AND amp media

06 August 2008 (W)

Talked to Jin last night about what to do with our non-cooperative sca2 and sca3. Got a new protocol for dealing with cotransformed sca2. Jin amplified fimH while he was still in lab...

The day - in order of occurence:

• 8:45a.m. - miniprepped two of our co-transformed sca2 samples for a new protocol (to rid co-transformation)
• UCSF BBQ
• 5:30p.m. - miniprepped the stock fimH that Jin amplified.-- want to send it to seq (check if fimH is really fimH...)
• diluted miniprepped, co-transformed sca2-7 sample 100x. Re-transformed onto CK plate.

Protocol for ridding co-transformation:

1. miniprep co-transformed sample
2. dilute sample 100x (0.5ul dna + 49.5ul water)
3. transform (with 15 min. rescue) into selected cell strain (as usual)
4. plate on double antibiotic plate
5. pick colonies the next day (maybe 2...)
6. streak on spec or amp plate and double-antibiotic plate to check for co-transformation
7. simultaneously put into media
8. miniprep the next day.

05 August 2008 (T)

• museum trip!
• came back, found that all 6 colonies of sca2 cotransformed on Amp plate...not good, not good..
• sequencing results: sca23 WORKED!!!! one assembly down! still 6 more (in Layer 1) to go...

sca3-9 is bad

Conclusion: I really want fimH to work...soon!!!!!

04 August 2008 (M)

Events of the day:

• seq results for sca2: the part was not there -- only the RFP was there!
• repicked 6 colonies for sca2 to check for cotransformation on an Amp plate.
• analyzed the gel that Bing ran yesterday - sca23-1, sca23-3, sca3-1,2,3, and 4.

The bands were kind of faint, and some were wrong, so we ran another gel, after miniprepping all our other sca3 and sca23 samples.

• 2nd digestion map: all 3 sca23 colonies looked good; sca3-9 looked best, and sca3-10 is our second choice for sequencing
• sent sca23-4 and sca3-9 for sequencing
• we decided to move everyone upstairs.

03 August 2008 (S)

Not in lab today, but Bing miniprepped and digestion mapped some of the samples from The One Block. I was very happy to hear that only one thing (one of the colonies of sca23) cotransformed.

02 August 2008 (Sa)

Came in at 11a.m.

Highlights

• of 5 colonies picked for sca2, none cotransformed, but colony 3 was red.
• Pelleted colony 1, 2, 4, and 5. Miniprepped colony 1 and 2.
• Digestion mapped colony 1 and 2. Bands were correct, but the single cut was really bright, for some unknown reason.
• Sent sca2-1 for sequencing.
• sca3 -- only about 10 colonies. Will pick all 10 -- grow in K/A media
• sca23 -- will pick 6 colonies -- grow in C/K media
• 24-well block shared with Bing:

Someone shall have the honor of miniprepping this block tomorrow...

01 August 2008 (F)

There was a lot more activity going today in the lab:

Statuses of our most recent endeavors:

Sca9:

• the two sca9 colonies that I picked yesterday were miniprepped
• digestion mapped, but looked really strange the first time (but Jin was still very positive about it...)
• digestion mapped 2nd time (using 2ul DNA) -- this time 4 bands showed up. Still not good
• We decided that we would use Aron's equivalent of sca9 (phoA!), which apparently worked perfectly fine for him.

HA tags

• Cici's sequenced <HA> and <HA! tags came relatively well...but are getting resequenced using g00101
• We went ahead and assembled sca23 (HA! + b1006)

Other sequencing news

• sca4 worked!!!
• but sca3 (our fimH that EVERYTHING needs to be assembled with) did not. Grrrrrrrrr...
• asked to have sca3 T1 resequenced using g00101 (we were worried there may be a mutation with ca998 -- many chromatagrams have come back with bases 70-80 very low signal...)
• Digestion mapped remaining sca3 colonies 2,3, and 4, hoping to send them to sequence.
• Digestion map for sca3-2,3, and 4 was WRONG... very wrong. We were expecting bands 1133 and 2362 bp: what we thought was the 1133 band was questionable, and there was no 2362 band!
• At 6:30p.m. we decided to re-re-transfer sca3, using Jin's incredibly awesome fimH (hopefully).

The world might as well come to an end if fimH doesn't work this time...

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