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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.
