Template:Team:UC Berkeley/Notebook/CC notes

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Revision as of 00:27, 27 June 2008

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.