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From 2013.igem.org

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Revision as of 13:33, 19 August 2013

{ "date" : "2013-07-01", "author" : "Gabriele", "title" : "Too many things in one single day", "content" : "

(1) PCR: SAM synthetase amplification

I amplified SAM synthetase by performing a PCR on the samples G1 and E2 from 17/06.

Sample	Quantification
G1	80ng/µl
E2	60ng/µl

The PCR was performed following the usual SAM extraction protocol.

PCR results were then run on a 1% agarose gel:

Since the gel shows two bands at nearly 1200bp, the PCR results are confirmed. The PCR products were then purified with the Promega kit.

Sample	Type	Quantity
G1	SAM synthetase	35.6ng/µl
E2	SAM synthetase	31.5ng/µl

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(2) pSB1A2+R0010+SAMsynthetase ligation screening

Then I screened the inocula from the 26/06 ligation. First I miniprepped the inocula:

The samples were then digested using the screening digestion protocol and then run on a 1% agarose gel.

Given that the gel shows only two bands (one at nearly 2kbp and the other at 200bp), SAM synthetase is not present and the ligation failed.

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(3) Linear pSB1C3 purification

I also purified with the usual protocol the linearized pSB1C3 from 19/06.

	Sample	Type	Quantity
G2	linear pSB1C3	74.4ng/µl
G3	linear pSB1C3	63.3ng/µl
G2A	linear pSB1C3	44.0ng/µl
G3A	linear pSB1C3	44.8ng/µl
G4A	linear pSB1C3	41.9ng/µl

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(4) O/N Digestion

Finally, I prepared an overnight digestion of the samples G1 (SAM synthetase) and G2 (linear pSB1C3) to try again the ligation tomorrow. I followed the usual protocol.", "tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3" }