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    2022-09-04
    • Notes
    • Metadata
    • Relevant Items
    • Level Ω GoldenBraid assembly (BsmBI -> pLX)
    • Qiagen QiaQuick PCR Purification Kit Protocol
    Editing disabled on read-only entry.

     
    2022-09-04

    Sunday, 9/4/2022Eluted DNA
    Sunday, 9/4/2022
    IN: 11:25 Sreenivas, Kalen, Richard
    OUT:
    Goal: PCR cleanup and assemble fragments obtained through PCR yesterday
    Using Qiagen QIAquick PCR Purification Kit (28106)
    also nanodropped synthetic spacer for concentration
    ●
    at 437 ng/uL
    ●
    serial dilute 1:200 to get 1.6uL at 2.185 ng/uL
    ●
    1:20 in TE and then 1:10 in ddH20
    update thermocylcer protocol for BsmBI-v2
    (42°C, 1.5 min → 16°C, 3 min) x 30 cycles → 60°C, 5 min → 80°C, 20 min
    ●
    incubate BsmBI-v2 at 42C (increase from 37C for BsaI)
    ○
    kit temperature at 42
    ○
    Activity at Temperature @37°C: 10%
    ●
    use Hi-T4 ligase or T4 ligase?
    ○
    Hi-T4
    ■
    not yet tested for golden gate assembly
    ■
    Incubate at 25°C
    ■
    Recommended Reaction Temp. (Useful Range)
    ●
    25°C (4-50°C)
    ○
    T4
    ■
    tested for golden gate assemby
    ■
    ligase used in protocol provided for golden gate BsmbI kit
    ■
    incubate at 16°C
    ■
    Recommended Reaction Temp. (Useful Range)
    ●
    25°C (4-37°C)
    ■
    expired in 01/22
    ●
    used expired T4 ligase
    in thermocycler at 3:00pm, should be done by 6:00pm

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