SBB12Ntbk-Huiting He

Huiting He 14:25, 16 February 2012 (EST)

==PCR product of Tar and PCA1 of Lz-AAAA-1==

===PCR product of Tar===
1. Obtain my oligos hhe0120F(32.6nM) and hhe0120R(32.9nM), dilute each oligo into 100uM by adding 326uL and 329uL ddH2O, respectively. <br>
2. Prepare 10uM of each oligo dilution by mixing
        9uL Water
        1uL 100uM oligo
3. label PCR tube "Tar", and add
        24uL ddH2O
        3.3uL 10x Expand Buffer "2"
        3.3uL dNTPs (2mM in each)
        1uL Oligo hhe0120F, 10uM
        1uL Oligo HHE0120R, 10uM
        0.5uL Template DNA "MG1655 Gen."
        0.5uL Expand polymerase "1"  (lastly)
4. Mix well and centrifuge briefly, then store on ice.

***The next steps (PCR amplification) are running by GSI:
     Note:first 10 cycles have shorter extensions than tha latter 20 cycles.
     Since the size of my predicted product is 802bp, which is under 2kb, we should use 2K55


===PCA1 product of Lz-AAAA-1===
1. label tube "Mix" and add 1uL of each oligo o1, o11, o12, then mix it well and centrifuge.<br>
2. label PCR tube "Lz-AAAA-1 PCA1", and add
     38 uL ddH2O
     5 ul 10x expand buffer
     5 ul 2mM dNTPs
     1 ul oligo mixture (100uM total, mixture of oligos from step 1)
     0.75 ul Expand polymerase
3. Mix well and centrifuge briefly, then store on ice.

***The next steps (JCA/PCA1 Program) are running by GSI:
      a) 2 min initial denature at 94oC
      b) 30 sec denature at 94oC
      c) 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
      d) 30 sec extension at 72oC
      e)repeat 2-4 30 times total

Karen D. Alvarez 16:57, 17 February 2012 (EST)

Analytical gel of PCR product(Tar) and PCA2

Analytical gel of PCR product(Tar)

1.label tube "tar-gel", mix 6uL Tar PCR product and 4uL dye