Template:Team:UC Berkeley/Notebook/CC notes

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Revision as of 22:24, 25 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 1 volume(since my DNA is 50 uL, so 1 volume is 50 uL) equivalent of Zymo ADB 
buffer to each reaction. Vortex to mix. 
2. Add 5 volumes of 95% ethanol (under fume hood, labeled flammable). Vortex to mix. 
The remaining steps are just like a normal Zymo clean-up reaction 
3. Label each Zymo column and fresh Eppendorf tubes with the appropriate names of your samples. 
4. Transfer your reaction mixtures into Zymo columns. Spin for 30 sec at full speed to pass the
 liquid through the column. 
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. This will dissolve any extra guanidinium chloride and salts sitting 
on the membrane 
6. Repeat the wash buffer step 3. Now nothing is present on the membrane but the DNA and a little 
ethanol and water 
7. Now spin the column for 90 sec at full speed to remove all traces of water/ethanol. 
8. Empty and discard the collection tube. Replace each collection tube with the appropriately-labeled fresh Eppendorf tubes. 
9. Add 90 ul of water directly to each of the membranes of the Zymo columns, and spin for 45 sec to elute the DNA into your fresh Eppendorf tubes.

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.