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PstI

PstI. T.Aquaticus genomic DNA pol I gene. DIYGem Starting point. PCR reaction #1. BB prefix + 5’ Taq pol I. PstI site. Mutagenic primer #1. PCR reaction #2. Mutagenic primer #2. PstI site. BB suffix + 3’ Taq pol I. Product of Reaction #1. 5’ Taq pol + BB prefix – PstI site.

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PstI

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  1. PstI T.Aquaticus genomic DNA pol I gene DIYGem Starting point

  2. PCR reaction #1 BB prefix + 5’ Taq pol I PstI site Mutagenic primer #1 PCR reaction #2 Mutagenic primer #2 PstI site BB suffix + 3’ Taq pol I Product of Reaction #1 5’ Taq pol + BB prefix – PstI site Product of Reaction #2 3’ Taq poI + BB suffix - Pst

  3. Step 2 3’ Taq PolI & BB suffix -PstI 5’ Taq PolI + BB prefix -PstI + Anneal Denature Denature Pfu Turbo extension

  4. Taq polI – Pst + BB prefix & suffix Clone, sequence, rejoice..

  5. Mutagenic primer 1 pstI pcr PET-Taq Mutagenic primer 2 DS pcr product Digest template with DpnI (cuts methylated DNA) Transform into cells Our “Plan B” using Strategene protocol to remove PstI site This plan took effect after several overlap extension failures.. Analyze colonies by pcr Attach BB prefix & suffix

  6. Taq Pol I -pst XbaI SpeI PstI EcoRI Poli Coli CMR Baltimore iGEM Team part

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