Template:Team:UC Berkeley/Notebook/AL notes

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Aronlau 18:33, 30 July 2008 (UTC)

7/26

I picked

7/25

On 7/25, I picked colonies for akl001, akl013, and akl015. I ran miniprep for ig17L. ig31L didn't grow. I sent AL74 out for sequencing. I started assembly for akl003 and akl005 and transfered ig31L again.

Aronlau 07:33, 25 July 2008 (UTC)

7/24

Today from 10-12, we had a minimeeting to discuss the progress of everyone. Everyone appears to be moving along at the same pace at the moment.

From 12-2, we discovered that the rbs.lambpp and rbs.phoApp actually had 2 extra bps, rendering them useless. All the parts that involved those parts were stored away in the -20 C freezer.

From 2-3, I ran a gel upstairs that included akl002, akl008, K112210 KC, Bca1092 AK, I716210 AC, Bca1107-C5 AC, K112608 AC, and Bca1126 CA.

UCB AL724.jpg

Lane Number Plasmid digested Result
1 akl002 C/K Good
2 akl002 C/K Good
3 akl002 C/K Good
4 akl008 AC didn't analyze
5 akl008 AC didn't analyze
6 akl008 AC didn't analyze
7 K112210 KC Good
8 K112210 KC Multiple Bands
9 Bca1092 AK Multiple Bands
10 Bca1092 AK Good
11 Ladder
12 I716210 AC Good
13 Bca1107-C5 AC Multiple Bands
14 Bca1107-C5 AC Good
15 K112608 AC didn't analyze
16 K112608 AC didn't analyze
17 Bca1126 CA Good
18 Bca1126 CA Good
19 Bca1126 CA Good



From 3-4 lunch. (Cashew Nut Chicken with rice significantly improves chances of getting good results on gels)

From 4-5, analyze the gel, which looks a good for the most part (thanks cashew nut chicken) and sent things off to be sequenced. Most importantly is that ftsQ was remade again today hopefully.

From 5-7, move ig17 and ig31 into lefty cells and clean tubes.

From 7-11:30, activated assembly machine (aka myself) and started assembling akl001, akl013, akl015.

Aronlau 07:44, 24 July 2008 (UTC)

7/16 Digestion Mapped (BamHI/XhoI)

ig126-wrong

ig152-good

ig64-wrong

ig17-good

ig15-good

ig129-good

ig135-good

ig144-wrong

ig157-good

ig160-good

ig121-good

7/15

Digestion Mapped (BamHI/XhoI)

ig126

ig152

ig64

ig17

ig15

ig129

ig135

ig144

ig157

ig160

ig121

Transfer Assembly K112607 A/C

I716210 A/C

Bca1107-C5 A/C

K112607 A/K

K112323 C/K

I716211 C/K

K112607 K/A

K112210 K/C

K112123 K/C

akl004 A/C

akl006 A/C

Grew up in culture ig20

ig31

ig85

7/14/2008

July 14 was a bright sunny day. But one thing that we noticed missing in lab was Jin. Jin, who was at the iGEM comp team program test, was gone for the morning and escaped the millions of questions that we had for him (or did he?). One thing for sure was that Sherine and I managed to picked the colonies for the plates that Molly worked so hard to prepare on Sunday. Because of the difficulty to properly miniprep like a machine (as the miniprep protocol was designed for a robot and we are not robots (just yet)), there were some issues getting desirable products. Therefore, we decided to scale back just a tiny bit to part production at the human level (somewhat dissapointed but what needs to be done has to be done). I was assigned the parts for protein purification and testing of prepro. I grew up some of the parts that were already made. My expectations was to finish the 5 layer assembly in 3 weeks.

Grew up the following in culture

ig126 ig152 ig64 ig17 ig15 ig129 ig135 ig144 ig157 ig160 ig121

7/13/2008 Although July 13 was a Sunday, the sight of the Golden Brick made it impossible not to go into lab. Using the part transfer protocol, I manage to do around 30 transfer ligations (Bing/Molly resposible for the rest of the work).


7/11/2008

On July 11, 2008, the iGEM began assembly of the transfer. Because of the low concentration of DNA, it is extremely difficult to analyze what is actually in our samples from digestion mapping, but also make assembly much less reliable. As uaual, I was the person responsible for ligation. I aliqoted 17.5 ul water, 2 ul T4 DNA Ligase Buffer and 0.5 ul T4 DNA Ligase to every well. Aside from that, it was mostly helping others around the lab. There was also people filming us today.

Aronlau 22:57, 10 July 2008 (UTC)

7/10/2008

A. Participated in the miniprepping assembly line.

B. Picked 3 colonies for H-NS in AC culture.


7/9/2008

A. For H-NS, Madhvi ran a gel upstairs for me. Afterward, I did zymo, digest, zymo, ligate, transformed into AC vector and plated.

B. I also ran a gel for Madhvi to see the digestion from digestion mapping, but there were no bands.


7/8/2008

A. H-NS oligo JH090 came today and started PCR.

B. Spend most of the time organizing spreadsheets for digestion mapping.

C. Picked colonies for parts that were made yesterday. Stock on appropriate stock plate and spec plate.


7/7/2008

A. Sorted the digestion plate data and performed ligation on the part transfer.

B. Grew up cultures for parts that need to be amplified for the future assembly.


7/6/2008

A. Ligation for the transfer of parts (refer to 7/6/2008 digestion plates for more information)

B. Colonies were picked and grown up in culture. Each were streaked on the appropriate antibiotic plates (A/C, A/K etc) and on spec plates. They were grown on both to check for co-transformation.


Aronlau 17:22, 6 July 2008 (UTC)

7/4/2008

A. Miniprepped H-NS and Pdps using magnetic beads and sent off to sequencing. Apparently the wells for miniprepping were somewhat small. To speed up the process, we would need bigger wells.

B. Tested assembly vectors Dirk pcred yesterday. They were ligated with an insert K112300 for AC/AK, K112301 for CA/CK and K112302 for KA/KC and plated on the appropriate plates.

C. Tested compotent cells (DH10B and Mach) that Jin made today. Plated compotetent cells on spec with pBca-1256 on spec and just cells on amp, cam, kam.


7/3/2008

A. We took assembly to a new level today. I did ligation for ~2.25 96 well plates.

B. Picked 3 colonies each for H-NS and Pdps and grew them up in LB with spec.


7/2/2008

A. Ran clonewell on the PCR products from yesterday. Bright bands for H-NS at 55 C and Pdps at 55 C, 45 C and with DMSO at 55 C. I used H-NS 55 C and Pdps 55 C for part making and stored the rest for future. I digested them, cleanup with zymo and transferred to assembly vector (H-NS/AC and Dps/CK). I also ligated, transformed and plated them.

B. I did ligation on the first 48 transfer of parts to assembly vectors.


7/1/2008

A. List of composite parts were being generated.

B. I ran PCR for H-NS and Pdps. The first time around it failed because too much loading dye was added to the gel. The second time around, we ran PCR for H-NS and Pdps at 55 C and 45 C and with and without DMSO.


Aronlau 08:15, 1 July 2008 (UTC)

6/30/2008

A. The list of composite parts is still being generated. Because the second batch of assembly vectors didn't look good on plates, I ran an E-Gel to see if there were any issues with the DNA. The size appeared to be correct.

B. My parts were transferred to the stock plates.

C. I did a ligation on the assembly vectors that Bing made and digested since I will be responsible for ligation. Bing did the transformation. We'll see how it goes tomorrow.

D. I helped Madhvi, Bing and Jin make compotent cells (just resuspension, aliquoting and capping (if this is even a task)).


6/29/2008

A. Sequencing data came back and 2 of the 3 colonies picked on Friday had perfect reads. This marks the end of basic parts making for me.


6/28/2008

A. Madhvi miniprepped my cultures and sent them off for sequencing.


6/27/2008

A. Results for resequencing came back. There was indeed a deletion in AL044. I picked 3 colonies for sequencing. I picked a 2 large colonies and 1 smaller one (maybe toxicity of holin can affect colony sizes).

B. Discussion on assembly line style asssembly. My part is to do ligation.


6/26/2008

A. Received sequencing for K112311. Thought that read AL044 contained a misread and told Richard to resequence.

B. The colonies for assembly vector plates were minimal. Jin showed us how to do colony pcr to screen for the insert but thermucycler broke down so couldn't really see the results.


6/25/2008

A. Minimeeting to discuss mostly about what we're doing and the next step we're taking.

B. Zymoclean up, digest assembly vectors, and ran gel. Bands on gel didn't look very bright. Bing restarted PCR. Also, digested insert, ligated and transform/plate.

C. 3 colonies for K112311 were miniprepped and sent out for sequencing.


Aronlau 17:51, 24 June 2008 (UTC)

6/24/2008

A. Received sequencing data. All perfect aside from K112311 with point mutation and K112316 with point mutation in EcoRI (possible poor signal so assumed it is perfect).

B. Picked 3 new colonies for K112311.

C. Set up PCR for assembly vectors.

6/23/2008

A. Sent out sample for sequencing

6/22/2008

A. Bing minipreped.


6/21/2008 A. Madhvi picked colonies for K112311, K112313, K112316 and K112319-1 and K112319-2.


6/20/2008 A. Clonewell to isoolate K112319.

B. Digestion and Zymocleanup

C. Ligation

D. Transformation

E. Religated and transformed for K112311, K112313, and K112316.


6/19/2008

A. Bing minipreped the colonies from yesterday and sent out for sequencing.

B. Perform PCR for K112319 A and B and separated with Clonewell. Started overlap extension.


6/18/2008

A. Received sequencing data. For K112319, there was a restriction site that was missed when designing oligos. Redesigned oligos. For parts with different sequences or point mutation, picked colonies.


6/17/2008

A. Bing minipreped and sent out for sequencing.

Aronlau 17:04, 17 June 2008 (UTC)

6/16/2008

A. For colonies with perfect match in sequencing, we made a stock of them on a plate.

1. Transformed the plasmids sent in for sequencing into compotent cells.
a. Mix of 220ul competent cells, 30ul KCM, 50ul water. Aliquot 30ul.
b. Add mix to 1ul of plasmid.
c. Incubate on ice for 10 min, heat shock at 42 C for 1.5 min, incubate on ice for 2 min.
d. Transfer to plate.

B. For colonies with point mutation, bad read, or other sequencing, we picked new colonies.


6/15/2008

The DNA sent for sequencing contained a lot of RNA, which affected some of the sequencing. Results for sequencing for pBca1256-K1112300 to pBca1256-K112320


6/14/2008

Bing came in to do a miniprep of the bacteria I picked yesterday using the Agencourt miniprep kit. This kit uses magnetic beads that bind dna to purify dna. The dna was sent out for sequencing analysis.


Aronlau 18:11, 13 June 2008 (UTC)

6/13/2008

A. Waited for colonies to grow larger.

B. Made 2xYT media and aliquoted 1ml.

C. Grew up the E.Coli in a 96 well plate.

6/12/2008

A. Use Clonewells to isolate DNA samples. Because sample B5 (K112312) may have been contaminated, PCR for b5 was done again.

B. Perform digestion using the follow procedure.

1. Make a solution of 5 ul NEB2, 15 ul DNA, 1 ul EcoRI, 1 ul BamHI, 28 ul water
2. Incubate for an hour

C. Clean up digestion with zymo columns.

1. Add 200ul ADB buffer to each of the digestion samples and pipette into zymo columns. Spin at 15s at full speed.
2. Add 200ul wash buffer. Spin for 15s at full speed.
3. Repeat step 2. Spin for an additional 90s at full speed.
4. Add 7ul of water to tube and spin for 30s at full speed.

D. Ligation

1. Mastermix: 6.5ul water, 1ul ligation buffer, 0.5 T4 DNA ligase, 1ul pBca1256. Aliquot 9ul.
2. Add 1ul of insert.
3. Cover with foil and incubate for 30 min at room temperature.

E. Transformation (always keep on ice)

1. 220ul competent cell in one tube, thaw on ice.
2. Add 30ul KCM and 20 ul water (both cold).
3. Invert ~2x to mix.
4. Aliquot 45ul cell into 10 ul of ligation rxn. (swirl and pipette up and down once)
5. Foil and Incubate 10 min on ice.
6. Heat shock 90s at 42 C.
7. Add 50 ul of LB.
8. Incubate 1 hr at 37 C.
9. Plate

Aronlau 22:08, 11 June 2008 (UTC)

6/9/2008

A. Filled out PCR table for parts K112300-K112321 to speed up PCR time when oligos arrive.


6/10/2008

Note: Oligos came and were diluted

A. Made the vector pBca1256

1. PCR-50 ul
a. 43 ul water, 1 ul 10mM dNTP, 5 ul 10x Expand Buffer, 1 ul 10uM ca1246F, 1ul 10uM ca1246R, 0.75 ul polymerase, 0.5 ul template (pBca1256)
2. Zymo cleanup
3. Digest vector using 1ul of EcoRI, BamHI, DpnI and 87 DNA/water solution. Jin ran a gel and said it is fine.


6/11/2008

A. PCR for parts K112300-K112321-First Try **Refer to construction files for oligos**

1. Added 5x more dNTPs, which competes for Mg in buffer with polymerase.
a. 18.5 ul water, 2.5 ul 10 mM dNTP, 1 ul buffer, 0.5 ul polyermase, 0.5 ul template, 1ul oligo 1, 1 ul oligo 2

B. PCR for parts K112300-K112321-Second Try

1. 20.375 ul water, 0.5 ul 10 mM dNTP, 1ul buffer, 0.375 ul polyermase, 0.25 ul template, 1ul oligo 1, 1ul oligo 2

Aronlau 22:00, 6 June 2008 (UTC)

6/2/2008 to 6/5/2008

A. Safety Training

B. Cloning Training-PCR, Purification, Digestion, Miniprep, Transformation, Autoclaving


6/4/2008 to 6/6/2008 - Design Oligos/Make Construction file

A. Oligos al0001 to al0024 for parts K112300-K112321

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