PCR and digestion controls

Aims

Since we have recently had problems with both PCR and restriction digests, we set up positive controls to ensure that there is nothing wrong with any of our enzymes or ...

Materials and Protocols

Please refer to the restriction digest, Phusion PCR and gel electrophoresis protocols.

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Results

Figure 1: Gel electrophoresis of the amplified PCR products and restriction digest

• Lane 1: 1 Kb ladder
• Lane 2: digested (linearised) pGFP-rrnB
• Lane 3: PCR product of pSB1C3
• Lane 4: PCR product of first fragment of rocF coding sequence
• Lane 5: 1 Kb ladder

Discussion

Worked correctly.. nothing is wrong with our enzymes.. in the case of our rocF plasmid PCRs, it must be the primers which are the problem.. new ones are supposed to be arriving today.. in the other cases, repeat..

yneA

Starch plating

Aims

Yesterday we replica plated Bacillus subtilis 168, that were transformed on Wednesday, onto starch plates. Today we aim to see whether or not the transformed Bacillus subtilis can breakdown starch. If they cannot break down starch there will be no halo when we test with iodine.

Materials

• Starch plates
• Pipette tips
• Iodine

Results

Unfortunately our colonies had halos. Although the insert has integrated, the colonies have antibiotic restistance, the insert has not integrated at the amyE locus.