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

Huiting He 00:53, 18 February 2012 (EST)

Analytical gel of PCR product(Tar) and PCA2 of Lz-AAAA-1
Analytical gel of PCR product(Tar)
1.Obtain my PCR product Tar
2.label tube "tar-gel", mix 6uL Tar PCR product Tar and 4uL dye, then load into the well(#8) of gel1 prepared by GSI. 

3.run the gel for about 30 mins, then take a picture.

PCA2 of Lz-AAAA-1
1. Obtain PCA1 product.
2. Since the product of PCA1 is expected small fragment (less than 300bp0, use small-frag Zymo cleanup procedure:
      a) Add 100 uL of Zymo ADB buffer (brown bottle) to the PCA1 tube.
      b)Transfer all liquid from PCA1 tube into the Zymo column 
      c)Add 500uL of Ethanol and pipette up and down to mix
      d)spin through 15 seconds, discard waste.
      e)Add 200 uL of Zymo Wash Buffer (white bottle which is basically 70% ethanol)
      f)spin through 15 seconds, discard waste.
      g)Add 200 uL of Zymo Wash Buffer
      h)spin through 15 seconds, discard waste.
      i)spin for 90 seconds, full speed to dry.
      j)add 50 uL water and elute with water (spin 60s) into a fresh Eppendorf tube [Note: the amount of water added depends on the starting amount of PCA product) 
  Now, the cleaned up assembly reaction as template for PCA2 is collected product into a fresh Eppendorf tube.
3. (Skip running on a gel due to smeary mess) Conduct amplification after zymo cleanup:
      a)label a PCR tube "PCA2 Lz-AAAA-1", then add:
               32.5 ul H2O
               5 ul 2mM dNTPs
               10 ul 5x phusion buffer
               1 ul purified pca product (from step 2)
               1 ul  outer oligo o1 (10 uM)
               1 ul  outer oligo o12 (10 uM)
               0.5 ul phusion
       b) Mix well and centrifuge briefly, then store on ice.

***The next steps (programming) are running by GSI:
             a) 2 min initial denature at 94°C
             b) 30 sec denature at 94oC
             c) 30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
             d) 30 sec extension at 68oC
             e) repeat 2-4 30 times total