Team:UNITN-Trento/Notebook/Labposts/07/02

From 2013.igem.org

{"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.
SampleQuantification
G180ng/µl
E260ng/µl

The PCR was performed following the usual SAM extraction protocol.
PCR mixes
G1 mixE2 mix
Template(50ng)0.63µl0.83µl
dNTPs0.5µl
primer Fw1µl
primer Rv1µl
buffer RBC5µl
Phusion pol0.3µl
RBC pol0.25µl
water41.32µl41.12µl

PCR results were then run on a 1% agarose gel:
Gel
Loading scheme
1kb ladderG1E2
\"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.
SampleTypeQuantity
G1SAM synthetase35.6ng/µl
E2SAM synthetase31.5ng/µl


(2) pSB1A2+R0010+SAMsynthetase ligation screening

Then I screened the inocula from the 26/06 ligation. First I miniprepped the inocula:
Miniprep quantities
1:1A490.9ng/µl
B252.1ng/µl
C345.6ng/µl
D295.3ng/µl
E315.6ng/µl
F226.2ng/µl
1:3A345.7ng/µl
B147.0ng/µl
C268.0ng/µl

The samples were then digested using the screening digestion protocol and then run on a 1% agarose gel.
Gel
Loading scheme
1kb ladder
1:1 A
1:1 B
1:1 C
1:1 D
1:1 E
1:1 F
1kb ladder
1:3 A
1:3 B
1:3 C
\"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.


(3) Linear pSB1C3 purification

I also purified with the usual protocol the linearized pSB1C3 from 19/06.
SampleTypeQuantity
G2linear pSB1C374.4ng/µl
G3linear pSB1C363.3ng/µl
G2Alinear pSB1C344.0ng/µl
G3Alinear pSB1C344.8ng/µl
G4Alinear pSB1C341.9ng/µl


(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.
Digestion mix
G1 SAMsynthetaseG2 linear pSB1C3
template (3µg)48.5µl40µl
XbaI2.5µl1.5µl
PstI2.5µl1.5µl
NEBuffer 210µl5µl
water26.5µl0
","tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3"}