Notebook : July 8

Summary:

FNR regulator system:

• performed another digestion for AmilCP, Pfnr plus sequence LacZ with Not I, Xho I, EcoR I and PST I.
• electrophoresis for those digest products
• enrichment culture for clone of the briobrick BBa_K1155000
• electrophoresis for extracted products of last friday

Lab work

Restriction digest

For some of the enzymes which are possible to use one common buffer, we did double digest

DNA	enzyme	buffer
AmilCP1	Not I	Orange
AmilCP2	Not I	Orange
AmilCP1	Xho I	Red
AmilCP2	Xho I	Red
AmilCP1	EcoR I	Orange
AmilCP2	EcoR I	Orange
LacZ1	EcoR I + PST I	Orange
LacZ2	EcoR I + PST I	Orange

DNA	2(µl)
buffer	2(µl)
Enzyme(foreach)	0.5(µl)
H20	15.5(µl)
total	20(µl)

The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).

Results:

Well 1,12 : marker Well 2: AmilCP1 + Ecor I Well 3: AmilCP2 + Ecor I Well 4: AmilCp1 + Not I Well 5: AmilCP2 + Not I Well 6: AmilCp1 + Xho I Well 7: AmilCp1 + Xho I Well 8: LacZ 1 + Ecor I + Pst I Well 9: LacZ 2 + Ecor I + Pst I Well 10: Nothing Well 11: fnr promoter gel 0.8%

Overnight incubation at 37°C with agitation.
The electrophoresis for the plasmids which we had extracted last week plus 3 other BioBrick promoter Bph R1, promoter Bph A1, Bph R2.

Volume added:

• Pfnr2: 10µl
• R1P: 10µl
• R2: 10µl
• A1P: 10µl

Migration at 100V for 25mins then continued till 45 minutes:
Results:

Well 1 : R2 Well 2 : R1P Well 3 : A1P Well 4 : Pfnr gel 0.8%

The concentration of Pfnr2 was detected by NanoDrop :

• C = 1788.8 ng/µl
• 260/280 = 2.08

We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.

Electrophoresis band size estimation

<p>We used Clonemanager for band size estimation:

Molecule	enzymes(Buffer)	Size
AmilCP clone	Nod I(Orange)	2046+693bp
AmilCP clone	XHOI(Red)	1842+892bp
BBa_K1155000	XHOI(Red)	1289+892bp
BBa_K1155000	ECoR I(Orange)	2151bp

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