SBB12Ntbk-Pamela Rios

Pamela Rios 16:23, 13 February 2012 (EST)

This is my next entry.

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Pamela Rios 14:23, 17 February 2012 (EST)

Part: SBB1202

Oligos: osbb1202F , osbb1202R Template: pBAD-T4L pcrpdt:916bp

1. Prepare 10 uM solution for each oligo

9uL Water 1uL 100uM oligo

2. Set up PCR (clonning)

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo ca998, 10uM
1uL Oligo osbb1333R, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

3. Store in ice

• PCR program run by GSI

Part: SBB1230

oligos: o1, o2, o10

1. Mix 1uL of each oligo (100uM)

2. Set up PCA (step1) for "Lz-AAAA-1 PCA1"

    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. store on ice

• PCA program run by GSI

      2 min initial denature at 94oC
     30 sec denature at 94oC
     30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
     30 sec extension at 72oC
     repeat 2-4 30 times total