Team:Cambridge/Lab book/Week 7

Monday (06/08/12)

PCR of Magnesium riboswitch vector fragment B and Magnesium promoter

Riboswitch and promoter gel. Lanes 2+3: Fragment B without 8 codon substitution, lanes 4+5: Fragment B with 8 codon substitution, lanes 6+7: magnesium sensitive promoter, lane 8: +ve control

• Normal PCR settings used, annealing temperature 57 °C, elongation step 90s long.

• Lane 5 accidentally loaded with a DNA ladder instead of loading dye.

• Expected fragment sizes:

• Lane 2-5: 3kbp

• Lane 6-7: 300bp

• After electrophoresis, found vector products had, for the most part, worked. Promoter elements were not amplified - no band of the expected size was observed.

• Positive control also failed, although this has ceased to work for several days, potentially due to DNA degradation.

Tuesday (07/08/12)

Production of electrocompetent e.coli

Gibson assembly of magnesium riboswitch and fluorescent construct

• NAD+ added to isothermal buffer*5 mix

• Gel slices from yesterday (of vector fragment B) purified.

• DNA added as follows:

• Without 8 codon substitution:

• Reaction 1: Tube 2 (07/08/12) (fragment B), Tube 1 (05/08/12) (fragment A), Tube 28 (29/08/12) (riboswitch DNA).

• Reaction 2: Tube 3 (07/08/12) (fragment B), Tube 2 (05/08/12) (fragment A), Tube 29 (29/08/12) (riboswitch DNA).

• With 8 codon substitution:

• Reaction 3: Tube 4 (07/08/12) (fragment B), Tube 1 (05/08/12) (fragment A), Tube 2 (18/07/12) (riboswitch DNA).

Wednesday (08/08/12)

Electrical transformation of competent e.coli with Gibson products

• Gibson constructs from yesterday (fluorescent and magnesium riboswitch) transformed into e.coli produced yesterday.

• Cells plated out onto 50 μg/ml ampicillin plates. Put in incubator overnight.

Thursday (09/08/12)

Making chemically competent e.coli

• 1l SOB made up.

PCR of mOrange vector

• Split mOrange vector amplification attempted again.

Friday (10/08/12)