Berk2010-Daniela

Daniela Mehech 18:03, 16 June 2010 (EDT)

Decided to redo all of B along with A. We can do them together on the robot by doubling everything but changing the source plate
Made dilution plate A, and added 60uL of dilution to dilution plate B. 7 with 11 still looked different
Distributed digestion cocktail to both dilution plates
Note: Robot begins pipetting air if it uses the same tip for two long. We stopped the program halfway through to change its tip
Distributed Righty and Lefty parts to reaction plates. Made sure there were duplicate columns for parts being transformed twice
Checked that at this point all wells had the same volume
Put plates in thermocycler to incubate and heat kill
Distributed antibiotics to 10 plates. All of LB agar was contaminated so we will pour that during the rescue step
added ligation mixture to each well and let incubate at room temperature
Began thawing cells (1 tube of each strain)
Poured agar plate
Skipped centrifuge step
Added cells to plate in two different concentrations (30uL, 70 uL)

Daniela Mehech 12:56, 16 June 2010 (EDT)

Robot 2AB assembly Stage 1

Yesterday we did stage 1 of our 2AB assembly only on plate B. Today we will repeat the process on plate A without making all of the mistakes that we did yesterday.

June 15:
Autoclaved our strips for plating. Learned how to work the autoclave machine

Made a new plate layout for the reaction plate. Wells are organized by what strain the part needs to be transformed into to
MISTAKE: Did not listen to Tim and organized wells by column instead of rows. Rows are better because the plating strips have 2 rows and 12 columns
MISTAKE: Did not read procedure all the way through in detail and assumed that we could use a well for two transformations. Our dilution and reaction plate needs to have duplicates of the parts that go into both Righty and Lefty strains. We have to add the duplicates in either the dilution or reaction plate
Made the dilution plate B (30ul of DNA into each well + 30ul of NEB2 stock diluted by a factor of 5). Dilution plate had the same layout as methylated stock plate so we did it by hand. Next time, we should consider adding duplicates of the parts being transformed twice.
NOTE: 7 with 11 was frozen in our methylated stocks plate. This is the part that was mini-prepped separately the day before. All other wells weren't frozen.

Had the robot make reaction plate (distribute digestion mastermix to all wells, distribute all Lefty parts, mix, distribute all righty parts, mix)
While the reaction plate incubated in the black thermocycler (which also did heat kill of the digestion enzymes) we distributed the antibiotics to the 24-well strips. We made antibiotics (dilute stock by 10, add 1ul of dye). This part went fine except we should have labeled the plates before we put them on the robot. Added LB agar to the plates and let them dry next to the flame.
MISTAKE: forgot to make second plate for parts being transformed twice. Column 5 in reaction plate needed to go to two different plates. We made the extra plate by hand

Had robot add the ligation cocktail to the wells and let it incubate at room temperature. We began thawing our bacteria.
Did transformation of bacteria. We had enough volume to seed at least 60uL of bacteria per column.
MISTAKE: Did not realize that the protocol was for seeding 10uL of bacteria. We wanted to seed about 150uL. We should not have done the centrifugation and aspiration of the supernatant step.
MISTAKE: ejecting tips from multi-channel before releasing cells on plate. We lost 60uL of our cells from column 7.
MISTAKE: We split the volume in column 5 into column 9 (because column 5 had to be transformed into both righty and lefty strains). Thus we also did not have enough volume for those parts.
MISTAKE: not taking a short break after lunch to re-energize and prevent making silly mistakes

To Do Today

• Run plate A and B together
• autoclave more 24-well strips
• Make dilution plate A and add volume to dilution plate B
• arrange reaction plate by rows instead of columns (have each row be transformed by the same strain)
• make more colored antibiotics
• label 24-well strip plates before putting them on robot and don't forget to make two plates for the parts being transformed twice
• do not centrifuge and aspirate supernatant after the recovery step

Daniela Mehech 20:37, 14 June 2010 (EDT)

Tim and Shelly picked colonies and mini-prepped for us over the weekend (Thank you!). We mini-prepped one colony (7 with 11), because nothing grew from our initial transformation. We also did RE mapping of 4 mini-preps to confirm that their tubes were labeled correctly (which they were).

Lane #	ID	Name	Expected fragments	'
1	4 with 10 #1	pMLL4-AC+B10sbb43	850	1990
2	4 with 10 #2	pMLL4-AC+B10sbb43	850	1990
3	4 with 18 #1	pMLL4-AC+B10sbb13	850	2160
4	4 with 18 #2	pMLL4-AC+B10sbb13	850	2160
5	4 with 9 #1	pMLL4-AC+B10Sbb20	850	2450
6	4 with 9 #2	pMLL4-AC+B10Sbb20	850	2450
7	9 with 3 #1	pMLL9-CA+Bjh1853	300	1500
8	9 with 3 #2	pMLL9-CA+Bjh1853	300	1500

Christoph and I began planning our Stage 1 assembly by writing a CSV file. We worked on replicating the spreadsheet Jennifer showed us to keep a detailed track of all of our parts.

To Do

Write final CSV file (both for making plates and for the digestion/ligation reaction plate) Make plates with proper antibiotics for plating Run Stage 1 on robot, transform, and plate

Daniela Mehech 19:04, 11 June 2010 (EDT)

Colonies grew from pMLL5+Bca1152 so I ran a colony PCR and inoculated 4 cultures. From colony PCR (band at 250), decided to miniprep colonies C and D. Sent D to sequencing, added it to parts plate (source) and transformation plate (destination). Mini prep from C went to Working Box

Added grown up Lefty and Righty strains to 500ml each of LB. Tim made competent cell stock for us

Finished with Christoph our CVS file for robot to dilute our parts in a new plate. (Note: when we were taking off the lids of the source plate, a few drops sprayed out. We dried the surface with a kimwipe, hopefully nothing got contaminated). Actually did the transformation by hand, but next time we should use the robot. Plated everything. Someone will move the plates from the incubator to the fridge tomorrow

To Do

Check that colonies grew in all of our transformation Get going on stage 1 of assembly

Daniela Mehech 14:38, 10 June 2010 (EDT)

Sequences for all parts we made were good (pMLL4+Bjh1882, pMLL5+Bca1091, pMLL6+Bjh2245)
Our competent cell cultures did not grow up at all - inoculated 2 more cultures of each strain and they appear to be growing well
Christoph and I double checked to confirm we had all the parts we need in the right vector - we're missing pMLL5+B10sbb37/Bca1152)
We took the part from the 140L stock, did a E/B digest, ligated it with pMLL5 digest and transformed it into jtk049 strain
I updated the pictures of the assembly tree
Began planning to do transformations of all our parts into righty and lefty strains

To Do

Pick colonies from Bca1152 transformation for colony PCR. Plate colonies in both CK plate and Spec plate. Nothing should grow in Spec plate
Make competent Pir cells
Transfer parts from working box to PCR plate
Write file for robot to do our transformations (first make dilutions from PCR plate)
When oligos come in - Biobrick upstream region of ef1-alpha

Daniela Mehech 15:33, 9 June 2010 (EDT)

For first Colony PCR of 1882 and 1091: Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)
For second Colony PCR of 1882 and 1091: Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)
Did all 4 mini-preps (stored in working box) and sent in pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing

Expected Colony PCR bands:

• pMLL4+bjh1882 = 849bps
• pMLL6+bjh2245 = 265bps
• pMLL5+bca1091 = 228bps
  

Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good-mini-preped lanes 2 and 3 and sent both for sequencing

Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
Designed three potential oligos for biobricking part Bjh2341CA (igemTen011, igemTen012, and igemTen013)
Bjh2341CA is a potential choano promoter (its a DNA region about 1kb upstream of ef1-alpha, a highly expressed gene)

TO DO

• Finish generating competent cells for "Lefty" and "Righty" strains.
• Analyze sequencing data for pMLL6+Bjh2245, pMLL4+Bjh1882 and pMLL5+Bca1091

Daniela Mehech 18:24, 8 June 2010 (EDT)

Still working with Christoph Did EB digest of pMLL6 and did gel purification. Did not take picture of gel because it looked right (a band showed up at approximately 2500)
Ligated pMLL6 digestion with Bjh2245 digestion from yesterday and tranformed into bacteria (not methylated strain, Jtk049)

Colonies appeared from our three transformations (Bjh 1882 with pMLL4and 2 samples of Bca1091 with pMLL5)
We ran a colony PCR on the 3 transformations but the results came out ambiguous (see picture below), so we picked 4 more colonies for more colony PCR
Lanes 1-4 are Bjh1882 and lanes 5-12 are Bca1091

Picked more colonies and ran second gel: Better results this time

To Do

• Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot. Restriction map for confirmation
• Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly. Possibly mini-prep if there is enough time

Daniela Mehech 18:58, 7 June 2010 (EDT)

Did the following with Christoph:
Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet)
Gel purified Bjh1882 and Bjh2245
Small fragment zymo cleanup of BCA1091
Couldn't see fragments. Turns out Bjh2245 is 97bp so we should have done a small zymo clean up instead of running it on a gel
Re-cut Bjh2245 and Bjh1882
Did a small zymo clean up of Bjh2245
Did zymo gel purification of Bjh1882 (see image below)

Next we ligated the digested part with the desired vector
Bjh1882+pMLL4 (AC)
Bjh2245+pMLL6 (KA)
Bca1091+pMLL5 (CK)

We don't have pMLL6 in digested form. We'll digest and ligate it tomorrow
We did transformation with Bjh1882+pMLL4 and Bca1091+pMLL5. We transformed them with a Lefty strain of E.Coli (Pir strain, yellow tube) so that the DNA is already methylated

To Do

Digest pMLL6 with Eco/Bam, ligate it with Bjh2245, transform check transformation of Bjh1882 and Bca1091