Team:Cambridge/Lab book/Week 6

Monday

PCR of vectors and mOrange

mOrange PCR run at multiple temperatures, every two lanes increacing the temperature of the annealing step by 2 °C. No lanes worked.

Top: Fusion, lux containing vector gel. Lanes 2-4, Vector DNA. Lane 5, +ve control. Lane 6, -ve control. Bottom: Vector gel. Lanes 1-3, Fluorescent contruct vector DNA. Lanes 4-6, Riboswitch construct vector DNA.

• Not all products were obtained during Friday's PCR. Most of these missing products were large vector backbones. They are being run again, with a much longer extension time of 300s. If that fails, primers will be ordered to split the vectors into manageable chunks, and the PCR reattempted when they arrive.

• PCR cycle x35:

• 15s Denaturing at 95 C
• 45s Annealing at 60 C
• 300s Extension at 72 C

• Remaining stray product had a slightly tricky secondary structure at the 3' end. It will be run at a series of annealing temperatures in a PCR machine capable of a temperature gradient.

• mOrange PCR run at many different temperatures, from 62 °C to 76 °C. Hopefully this should resolve the difficulties we have been having.

Separation of vector and mOrange DNA

• Positive control produced no band. No primer smear - primers may not be in mix for some reason.

• Realized correct lux vector template was not added during PCR preparation, consequently no amplification occured. Still appears to be a primer smear.

• Fluorescent construct produced several bands. Appears to be due to mis-priming during PCR. Either changing the primers or raising the annealing temperature should solve this problem, but may mean that we have to do this PCR separately.

• Riboswitch vector also failed. However, the extraction from the previous PCR run may have worked. We will try producing a functional plasmid with this extraction before running this PCR again.

Construction of riboswitch plasmid with Gibson Assembly

• DNA from lanes 27+28, 27+29, 27+30 from gels run on Friday fused together with Gibson assembly to produce riboswitch construct. This does not have replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch.

• DNA from lanes 22,23 and 24 fused with riboswitch DNA produced two weeks ago to produce riboswitch construct. This has replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch.

Transformation of Bacillus with riboswitch construct

• Plasmids made by Gibson transformed into bacillus cells made two weeks ago and transformants plated out on 5μg/ml chloramphenicol plates.

Tuesday

New biobricks

• E.coli containing biobricks ordered were plated out on kanomycin plates (50μg/ml).

Transformation of Bacillus with riboswitch construct

• Plasmids made by Gibson transformed into bacillus cells made two weeks ago and transformants plated out on 5μg/ml chloramphenicol plates.

Transformation of E.coli with riboswitch construct

• Plasmids made by Gibson transformed into TOP10 e.coli cells and transformants plated out on 100μg/ml ampicillin plates.

Wednesday

Mg2+ Riboswitch

• Successful colonies produced from transformations two days ago streaked out onto chloramphenicol (5 μg/ml) containing plates.

• Colonies also grown up in 10ml of medium A for use with plate reader later.

PCR of vector DNA

• Standard PCR rerun, this time splitting fluorescent vector into two separate sections, one of 3kb and one of 4.5kb.

• New primers used:

• Fragment 1 reverse:

• Fragment 2 forward:

Thursday

Friday

PCR of split Mg2+ vector

results of split magnesium vector PCR. None of the products worked

• PCR of magnesium riboswitch vector repeated with primers to split plasmid (kindly provided by P.J.Steiner).

• Lanes 2 + 3: Fragment A (center - cut site (promotor side))

• Lanes 4 + 5: Fragment B (without 8 codon substitution) (cut site (lac I side) - center)

• Lanes 6 + 7: Fragment B (with 8 codon substitution) (cut stie (lac I side) - center)

• Gels run, found PCR was unsuccessful.