Team:UNITN-Trento/Notebook/Labposts/07/10

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Revision as of 07:22, 9 September 2013

{ "date" : "2013-07-25", "author" : "gabriele", "title" : "Screening - GOT IT! F**k yeah!", "content" : "I checked yesterday's inocula and, surprisingly and sadly, 4 out of 7 were red!!! I must admit that I don't get how it is possible to have red inocula when I treated the linearized-by-PCR pSB1C3 with the DpnI enzyme... that's crazy... anyway the quantification results are the following:

Inocula	Quantity
ON 1:1 A	RED
ON 1:1 B	RED
ON 1:1 C	216.4ng/µl
ON 1:1 D	215.5ng/µl
ON 1:1 E	RED
Short 1:1	RED
Short 1:3	315.5ng/µl

Screening digestion

Screening was performed with BamHI-HF and PstI-HF. While PstI-HF cuts at the suffix (the end of the insert), BamHI-HF is able to cut only SAMsynthetase at 102th base position. So, the screening digestion will show one band at nearly 3100bp if pSB1C3 contains Plac+RFP, at nearly 2000bp if an 'empty' pSB1C3 is present and two bands (one at nearly 2200bp and the other at nearly 1000bp) if we have pSB1C3 with SAMsynthetase.

Digestion mixes

	11C	11D	13S
template	4.6µl	3.17µl
PstI-HF	1µl
BamHI-HF
NEBuffer 4	2µl
BSA
Water	9.4µl	10.83µl

The screening digestions were incubated at 37°C for 1 hour and the run on a 1% agarose gel.

Gel

Loading scheme
1kb ladder	11C	11D	13S

$\"Gel\"$

As you can see, the gel shows a band at nearly 2kbp and one at nearly 1kbp at the 13S lane, so that sample should contain the desired construct (pSB1C3+SAMsynthetase).", "tags" : "SAMsynthetase" }