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 - 58 °C - 30 seconds

• Elongation - 72 °C - 30 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.

PCR of PJS130 vector for Mg riboswitch construct

Gel from amplification of PJS130 vector for MGRS construct. Lanes 2 + 3: Fragment A. Lanes 4 + 5: Fragment B with 8 codon substitution. Lanes 6 + 7: Fragment B without 8 codon substitution.

• Cycle settings:

• Melting - 98 °C - 10 seconds

• Annealing - 60 °C - 30 seconds

• Elongation - 72 °C - 110 seconds

• Fragment of correct size produced in lane 5, but too faint to be extracted successfully. None of the other lanes were successful. We will try this again tomorrow, at 58 °C and with 35 cycles (as is usual) instead of 30.

• Note that phusion enzyme was left with primers and template DNA for about half an hour before reaction began, possibly causing degradation of the primer DNA.

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

• 20 μl of Gibson reaction mix transformed into e.coli cells. Transformants plated out onto 100 μg/ml ampicillin plates.

Saturday (01/09/12)

Sunday (02/09/12)