549 pET28b-Phi29DNAPol
4/8/2012
Gene synthesis of 549 with Kapa Hi Fi, 25 cycles
Fragment 1: using oligo mix 1-1 to 1-26, using 1-1 & 1-26 primers
Fragment 2: using oligo mix 2-1 to 2-27, using 2-1 & 2-27 primers
4/8/2012: Fragment 2 gene synthesis
cut out band and reamplify.
4/10/2012
Gibson Assembly of fragment 1 + 2 and vector. Use 5 ul mixture with enzyme mix (Gibson or SystemBio)
Lane 1: fragment 1 + Fragment 2 + pET28b, no Gibson mix is added
Lane 2: fragment 1 + Fragment 2 + pET28b, use systembio reagent
Lane 3: fragment 1 + Fragment 2 + pET28b, add 15 ul Gibson Mix.
Cut out bands, gel purify and transform Top10.
Cloning of 549_pET28b-Phi29DNAPol
[edit]Purpose: Cloning of 549_pET28b-Phi29DNAPol
Date:Dec, 2012
Notebook:
Page:
Clone ID: 549
Experimenters: shanyi
Results
[edit]Cloning
[edit]gene synthesis 2 fragments.
join 2 fragments by PCR
digest pET28b WITH Nco1+ Xho1
Colony Screening
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Result intepretation/Summary
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Conclusion
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