Template:Team:UC Berkeley/Notebook/CC notes

From 2008.igem.org

Revision as of 01:55, 23 July 2008

Cicikashou 01:48, 23 July 2008 (UTC)

I'll need to write up my notes everday from now!!!

So today I checked the sequencing results for CC9-12. Somehow I ordered forward and reverse sequencing for CC9 and CC10. And I fixed for CC11 CC12 to only forward sequencing. I made a mistake in puting fimH> into AC media, but it should actually be KA. So we'll be minipreping and start the assembly tomorrow. The sequencing result for sca16 and sca20 is bad read, so we'll re-assembly them tomorrow.

CC number     SCA number         result       
CC9           Sca15              bad read-re-assembly
CC10          Sca17              bad read-re-assembly
CC11          Sca18              bad read-re-sequencing
CC12          Sca19              bad read-re-sequencing

Tomorrow we'll re-assembly sca15,16,17, and 20. Depend on how sca11 and sca12 sequencing result will be, we might want to assemly them.

Today we re-assembly the sca15 and sca21 (all 10 colonies are co-transformed), during the zymol clean up , we forgot to put the column into 1.5mL tube after eliuting with water, so we redo them again!!! And duing the ligation I mislabeled two lefty for sca21, but we continued anyways. I think I'll redo them tomorrow just to make sure.

Cicikashou 01:55, 23 July 2008 (UTC)

today I checked for co-transformed on ACK plate for sca16,20,21-25. sca21 has all 5 colonies cotransformed, so we minipreped rest of them and send out for sequencing. Sherine picked 10 more colonies today for sca21.

Cicikashou 16 July 2008 (UTC)

This morning I checked on the antibiotic/spec plates for <AP>, no colonies grow on spec, so I picked single colony and grow in media. I also picked up the three <HA!,Pbad,rbs1-A that needed to be minipreped. I did minipreping for all three of them. And in the afternoon we did digestion mapping and the results all came out good. So Pbad,all the tag parts wo stop codon,rbs1-A, and fimH are put to first layer assembly.Lefty digestion are pbad and fimH, and rest of them all are digested with Bgl/Xhol. Aron did the assembly for us, and transformed pbad.rbs1-A into lefty cells, rest into righty cells. I sent out an email today to Richard since I still haven't got my sequencing results back. When I checked the sequencing, rbsbarstar is perfect read. But phoA only got perfect read around 820bp,which is normal. Sin phoA is around 1200bp, I need to do a reverse seuencing run and check the rest of the sequence. Hopefully it will turn out all right and all ready to go into assembly.

Cicikashou 15 July 2008 (UTC)

This morning we minipreped what we cultured last night, and did digestion mapping for these samples. <AP> need to be restreaked from the pr plate since the mapping result is not correct. We also pick colonies from the stock plates for three more samples.

Cicikashou 14 July 2008 (UTC)

Today Molly walked me through the transformation for transfer and I'll be doing it tomorrow. This morning I went to the comp team testing meeting and tried out the clotho the comp team designed. We also have a meeting regarding to our current assembly line method. So the new idea is that we'll each be doing individual mini assembly, and everyone's in charge of some projects. So me and Sherine will be doing the tag project, and since our task is lighter than others, we'll also be doing the large assembly also. Tonight we looked at the composit parts we are suppose to make, find their stock location on the plate, and grow up the colonies. I left a little early tonight so Sherine did the picking for me I suppose.

Cicikashou 9 July 2008 (UTC)

This morning I praticed for my presentation. I didn't prepared that well, but oh well, it's ok. Today is extremely hot!!!! I came back to lab around two o'clock and have been working on my notes since then.

Cicikashou 8 July 2008 (UTC)

The oligos came in and we did PCR yester day, so taday I mainly working on my basic parts. I did a gel clean up--all my bands show up really well. Then I did digestion, zymol clean-up, ligation, and transformation. I didn't do the the plating for the transformation because mom wanted to leave early. I screened some more colnies again, and I didn't get to do the colony PCR! I asked Shrine to help me with the colony PCR and do the plating for me too.

Cicikashou 7 July 2008 (UTC)

Today I was working with Madhvi on screening the colonies, doing colony PCR and running the gel. This takes really long time because we spent long time making spreadsheet and deciding which colony to do PCR. We pick 22 and used the toothpick to pick colonies. Apprently the toothpicks obsorb liquid so by the end when we were ready to do PCR, not much liquid was left in the PCR tubes! So we re-did the PCR and used the pipett tips to pick colonies. And in the afternoon we ran a gel and the results didn't look so good. A lot of them didn't showing bands. I left lab at nine o'clock, I was tired!

Cicikashou 6 July 2008 (UTC)

So taday I finally installed the Illustrator and tried to work on my design! I figured that by tracing the text using tool box is too painful for me and it didn't really look good. So I'll draw more details for each letter and scan it and do a live scan on Illustrator. So the result looks ok, I just need to work on it to make it better.

Cicikashou 19:27, 3 July 2008 (UTC)

This morning I hurried and finished drawing the logo on bigger paper. I went up to meet Kelvin around 10:30 and Kevin tried to come down and meet me. But I guessed we both missed each other! So Kevin scaned the two drawings and showed me how to play around the Illustrator. This morning I picked some colonies and incubated them in different culture media (AK,CK,AC, etc). So I'll see what else I'll do to help today!

Cicikashou 19:27, 2 July 2008 (UTC)

I did a lot today. In the morning I redesigned the basic parts I was assigned. One part was thrown out, so now I have three parts to make. Most of the afternoon I was doing minipreping. I want to make sure I was doing it right so I didn't hurry. I finished minipreping round two thirty and help to find the ig number in the assembly tree. So I ate my lunch around three! I guessed we finally starting the assembly today! Yeah!

Cicikashou 23:23, 1 July 2008 (UTC)

So today we came in really early and we spent most of the morning helping to do the assemply tree. We were trying to find the part number for each part, and the spreadsheet has been updated for about four times, so we were trying to keep up with the update! I need to remind myself that on Thurdady morning I'll need to go to Kate's office and learn about how to use the Illustrator for doing the Lysophonix text. Which means that I need to scan the pic that I designed. Around five o'clock Jin assigned us some parts, so we spent one hour to design the oligos.

Cicikashou 17:46, 30 June 2008 (UTC)

On Sunday evening I checked the DNA sequencing result for my parts. They all look fine except that for CC 006 and CC006 they switch with the plasmid and I have no idea what happen. Did I made a mistake during the lab or did I made mistake while loadaing the DAN to the PCR tube that sent out for sequencing?

Cicikashou 20:09, 27 June 2008 (UTC)

Today I did the miniprep, so some accidents happened when I was doing the lab, I forgot to screw the centrifuge cap, which makes the centrifuge make a lot of noise, and I accidentally drop the column to the counter, and all the liquid just spill! So I need to be carefully next time. We are also helping to do a analitical gel after Molly's colony PCR. It's different than clone-well. The DAN ladder has 10uL laddr and 10uLwater. and it runs for 20 min.

miniprep

1. Pellet 1.5 mL saturated culture by spinning full speed for 30 sec. 
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL) 
3. Add 250 ul of P1 buffer into each tube. Vortex well to resuspend cells.
   You should not see anything solid at the bottom of the tube 
4. Add 250 ul of P2 buffer (a base that denature everything and causes cells to lyse).
   Gently invert until uniformly light blue. 
5. Add 350 ul of N3 buffer (an acid of pH ~5 that cuases cell junk, including protein
   and chromosomal DNA, to 6. precipitate and leaves plasmids and other small molecules
   in solution). Slowly invert ten times. 
6. Spin in centrifuge at top speed for 5 min. 
7. Label blue columns with an alcohol-resistant lab pen. 
8. Pour liquid into columns, and place the columns into the centrifuge.
   Spin at max speed for 30 sec. 
9. Dump liquid out of the collection tubes into waste container. 
10. Wash each column with 500 ul of PB buffer. 
11. Spin in centrifuge for approximately 15 sec, then flick out the liquid again. 
12. Wash with 750 ul of PE buffer. This washes the salts off the resins 
13. Spin in centrifuge for approximately 15 sec, then flick out the liquid again. 
14. Spin one more time at max speed for 90 sec to dry out resin. 
14. Label new tubes and put columns in them. Discard collection tubes. 
16. Elute them by squirting 50 ul of water directly onto the resin in each column.
    Get as close to the resin with your pipette tip as possible without touching it 
17. Spin in centrifuge at top speed for 30 sec. 
18. Take out columns and cap the tubes.

Cicikashou 00:33, 26 June 2008 (UTC)

So last night we went home like 8PM to incubate the bacteria, so today we only need to check the plates and incubate the colonies. I picked 2 colonies for each plate, prepared the stocke plate, and incubate the tubes in the room. And we did a miniprep today, so tomorrow the lab should not be hard. And I'm in charge of designing our title to put on the wiki, and Sherine helped me with me too. So I'll rescetch and scan it and upload it.

Cicikashou 22:24, 25 June 2008 (UTC)

This morning we were suppose to have a mini-meeting at 10 AM, and I totally forgot and I was like half an hour late!!! I felt really bad about it, so from now on I should be getting up a little bit earlier and come to lab at 10. I would record the notes I took from mini-meeting later, but for now I'll record what I've done for lab so far. So the PCR was ready when I return to lab, and we were doing the zymol clean up for the wobble PCR.

Clean-up with Zymo Columns

Products Between 20 and 300 bp

1. Add 50 uL (since it's 1 volume of the 50 uL DNA)of  Zymo ADB buffer to each reaction. Vortex to mix. 
2. Add 250 uL of 95% ethanol. Pipettet up and down to mix.
3. Label your columns and tubes. 
4. Transfer your all mixtures into Zymo columns. Spin for 30 sec 
5. Empty collection tubes and put back onto the columns. Wash with 200 ul wash buffer and again 
spin for 30 sec at full speed. 
6. Now spin the column for 90 sec at full speed to remove all traces of water/ethanol. 
7. Empty and discard the collection tube. Replace each collection tube with 1.5 uL tubes
8. Add 90 ul of water directly to each of the membranes of the Zymo columns, and spin for 90 sec to 
elute the DNA into your fresh Eppendorf tubes. 

Restriction Digest (total amount of 100 uL)

88 uL DNA and Water
10 uL of NEB Buffer 2
1 uL EcoRI
1 uL BamHI

vortex and spin to mix
incubate for 1hr

Clean up Digest

After 1hr,
repeat the zymo clean-up, but elute with 10uL water.

This procedure will:
remove the leftover buffer
remove the restriction enzymes
remove the short tails liberated by digestion
concentrate the DNA

Mastermix: 6.5 ul water
           1 ul ligation buffer
           0.5 T4 DNA ligase
           1 ul pBca1256 digested  
Aliquot 9 ul into fresh tubes. 
Add 1 ul of your insert. 
Cover with foil and incubate for 30 min at room temperature. 

Transformation

**** while waiting for the ligation, make sure to take out the competent cells! *****

1. Retrieve your competent cells from the -80 (on the third shelf) and thaw on ice. 
2. Add 30 ul cold KCM and 20 ul cold water to each tube of competent cells. 
   Invert ~2x to mix. 
3. Take the top of an empty pipette tip box, add ice and water.
   Do NOT let any water get into the tubes.
4. Combine 45 ul of your cells into 10 ul of your ligation rxn while
   remembering to keep all tubes on ice. pipette up and down once to mix. 
5. Foil all tubes and incubate 10 min in ice-water. 
6. Heat shock your cells by placing them in a 42 C water bath for 90 sec. 
7. Remove and incubate on ice for 2 min. 
8. Rub ethanol/flame top of foil to sterilize. Add 50 ul of LB 2YT to each
   tube by poking holes through the foil with your pipette tips. 
   Re-cover all tubes with foil and incubate at 37 C for 1 hour. 
Plate using the sterilized glass-bead method or with standard spreaders. 

Cicikashou 19:41, 24 June 2008 (UTC)

This morning I was working on recording things, so that's what I did: I made up the construction files, I remade my oligo/part on google doc (somehow it crashes the first time), and I wrote up my notes. So things should be set up for me now. And since I'm waiting for the oligos to come and do the wobble PCR, I'll just writ up the protocol:

For deluting the oligos: since it comes in solid, you must resuspend it in water to a final concentration of 100uM (uM is 10^-6 molar). Since IDT measures how much moles of DNA are present in the tube and writes it on the tube.

Let's say it's 23.4 nmoles of material, you therefore add 234uL of water to the tube.
To set up PCR, you'll want to use 10uM stocks, so do a 10x dilution of the oligos.
To achieve this, add 1uL of 100uM oligo to 9uL of water, then mix it. 

'''Wobble PCR (50ul)'''

Mastermix: 40 ul water
           1.5 ul MgCl2
           5 ul buffer (Taq)
           1 ul 10mM dNTP
           0.5 ul Taq Polymerase 

Aliquot 48 ul into each tube 
Add 1 ul 100 uM Oligo 1, 1 ul 100 uM Oligo 2. 
Load into thermocycler. 
Select the program WOBBLE55 or WOBBLE45 and run. 

*** THINGS I LEARNED TODAY****
Because we are doing the wobble PCR, we don't need to delute the oligos for the second time. 
The idea of deluting the oligos is to have less concentration of oligos when doing the PCR, 
but for wobble, since we are using oligos for amplification without templates, we need a lot 
of oligos, so the 100uM is fine.

this is how much water is added to each oligo:
               nmol of oligo                    mL of water added
cc001         35.9                              359
cc002         26.1                              261
cc003         29.3                              293
cc004         30.4                              304
cc005         30.5                              305 
cc006         37.5                              375    

THINGS I LEARNED TODAY And we used the wobble 55 program for PCR. The 55 has a higher annealing temperature, which is suppose to make oligo bind more specific to annealing/overlap part, thus it allows the PCR product to be more pure, in contrast, the 44 program had lower annealing tempature which allows more oligo binding but might not be as accurate.

23 June 2008

So our assignment was to collect the HA, MYC, FLAG, and AP tag sequence found in vector that can be used in E.coli. I found the MYC tag. Because they are under 30bp, we decide to do EIPCR and the whole morning me and Sherine were trying to understand EIPCR and design our oligos. We looked at the turtorial and ask Dirk and Terry about it, but I was still pretty confused because I didn't get where we should put the restricition sites and how it will work. And then Jin send us the annealing regions on the pBca1256 which causes more confusion. So for the forward oligo, it's

insert-EcoRI-insert-Bgl-[part]-Bam-annealing region

The annealing reigon starts from the Bam site and it's about 20bp. The reverse oligo is the reverse ca1246 that Jin sent, so basically we are using the same reverse oligo evertime. I still need to do the EIPCR on Ape for more practice. But the thing is that later Jin decided that since the oligos are too long, we are going to do the wobble! So how we design the oligos is that we got the sequence:

insert-EcoRI-insert-Bgl-[part]-Bam-insert

and there should be 20bp overlap and GC should be b/ 40-50%. And I record my oligos to google doc and did my construction files.

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