Team:Cambridge/Lab book/Week 10

Monday (27/08/12)

Tuesday (28/08/12)

Wednesday (29/08/12)

Thursday (30/08/12)

PCR of Mg2+ riboswitch from genomic DNA

Gel from amplification of the riboswitch DNA. Lanes 2 + 3: with 8 codon substitution. Lanes 4 + 5: without 8 codon substitution.

• Cycle settings:

• Melting - 98 °C - 10 seconds

• Annealing - 60 °C - 30 seconds

• Elongation - 72 °C - 110 seconds

• Fragments of correct size produced for all except lane 5 produced. In this lane, DNA appears to have accumulated in the well, indicating it may be genomic. In future, will use lower numbers of template cells to avoid getting so much genomic DNA.

• Products extracted and purified.

Friday (31/08/12)

PCR of PJS130 vector for MgRS construct

Gel from amplification of PJS130 fragments. Lanes 2 + 3: Fragment A. Lanes 4 + 5: Fragment B with eight codon substitution. Lanes 6 + 7: Fragment B without eight codon substitution. Lane 8: Negative control.

• Cycle settings:

• Melting - 98 °C - 10 seconds

• Annealing - 58 °C - 30 seconds

• Elongation - 72 °C - 120 seconds

• Products of correct sizes (5.5kbp and 3.5 kbp) produced for all reactions, although lanes 2 and 4 failed to produce any product, despite primer smear. Most likely, template was not added, or one of the primers was not added.

• Products excised and purified.

Gibson assembly of Mg2+ riboswitch construct

• Reaction 1: Without 8 codon substitution: Vec A, Vec B -8 (replicate 1), Genomic -8.

• Reaction 2: Without 8 codon substitution: Vec A, Vec B -8 (replicate 2), Genomic -8.

• Reaction 3: With 8 codon substitution: Vec A, Vec B +8, Genomic +8 (replicate 1).

• Reaction 4: With 8 codon substitution: Vec A, Vec B +8, Genomic +8 (replicate 2).

Transformation of e.coli with Gibson products

Saturday (01/09/12)

Sunday (02/09/12)