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From 2013.igem.org

Contents 1 Isolate plasmid from overnight culture 2 Glycerol stocks 3 Gel Purification of the pGEM-T pasmid double digestions ran in the 11/09/13 4 Digestion of the samples purified with the enzyme PstI-HF and XbaI 5 Double digestion of the isolated pGEM plasmids ligated with the TOD operon genes (X, F and TOBG) 5.1 Master Mix of the PstI/XbaI double digestion

Isolate plasmid from overnight culture

• Using Omega mini prep kit protocol
• Samples:
  • TODF 1,2,3,
  • TODX 1, 2, 3
  • TOBG 1, 2, 3,
  • RFP 1, 2, 3

sample	Volume (ul)	Concentration (ng/ul)
TODF1	43	120
TODF2	43	98.8
TODX1	42	399.3
TODX2	44	442.2
TODX3	43	417.5
TOBG1	43	262.4
TOBG2	47	319.2
TOBG3	41	338.4
RFP1	41	77.7
RFP2	41	32.7
RFP3	42	59

Glycerol stocks

• Took 750ul from overnight culture and centrifuged at full speed for ~2 minutes
• Supernatant was removed
• Pellet resuspended in 375ul of HMFM
• Samples were then frozen at -80

Gel Purification of the pGEM-T pasmid double digestions ran in the 11/09/13

The ge was purified by using the The Zymoclean Gel DNA recovery Kit and its protocol was followed.

Nanodrop Results

Sample	Volume	Concentration (ng/ul)	280/260	260/230
TODX	48.5	7.1	1.83	0.72
TODF	45.5	21.7	1.89	1.19
TOBG	47.7	9	1.77	0.70
pSB1C3 (RFP)	46.2	7.3	1.81	0.16

Digestion of the samples purified with the enzyme PstI-HF and XbaI

• Two single digestions were set up to test if one of the enzymes was not working properly.

DNA volume	Volume	Cutsmart buffer	Enzyme volume	dH2O added to total volume of 60 ul
TODX	23	6	1	30
TODF	22.5	6	1	30.5
TOBG	22	6	1	31
pSB1C3 (RFP)	20.5	6	1	32.5

Gel order: TODX, TODX, TODF, TODF, TOBG, TOBG, pSB1C3, pSB1C3 (XbaI); TODX, TODF, TOBG, pSB1C3 (PstI)

The picture above shows that the assumption that one of the enzymes did not working properly was wrong, as it shows the same result as the gel run on the 11/09/13.

Double digestion of the isolated pGEM plasmids ligated with the TOD operon genes (X, F and TOBG)

• As the digestions with the plasmids that were stored in the freezer did not work new digestins were set up with the isolated plasmids from the overnight culture.
• The pGEM-T plasmid contain the restrictions sites for PstI and SpeI, which are also present in the amplified TOD operon gene and the pSB1C3 plasmid.
• To avoid the recircularization of the pSB1C3 backbone when ligating the genes with this plasmid, the double digestion was set up with the enzymes PstI and XbaI.
• Another double digestion with EcoRI and SpeI was set up to test if the restrictions sites present in the TOD genes are not mutated. If the bands in the gel shows a linear band, it can be assumed that the enzymes cut the pGEM plasmids in its PstI and SpeI restriction sites.

Master Mix of the PstI/XbaI double digestion