Team:UNITN-Trento/Notebook/Labposts/07/20

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{ "date" : "2013-07-10", "author" : "gabriele-viola", "title" : "Reboot: starting from the beginning", "content" : "Since the new forward primer (with the entire prefix) arrived, I started again from the beginning.

SAM synthetase extraction

The new primer forward has just the EcoRI restriction site added at its beginning (complete prefix):
GAATTCGCGGCCGCTTCTAGAGAAGGAGGAACTACTATGGCAAAACACCTTTTT
It has a Tm = 68.7°C.

Then I performed two Phusion PCR (one GC and one HF) and one Phusion/RBC PCR to identify the best protocol for SAM synthetase extraction with the new primer.
Phusion PCRs
PCR Mixes
Mix HF (1)Mix GC (2)
Phusion Buffer HF10µl0
Phusion Buffer GC010µl
dNTPs1µl
template1µl
Primer Fw2.5µl
Primer Rv
Phusion pol0.5µl
Water33.5µl


Phusion Settings
StepTemperatureTimeGo to
198°C30 sec
298°C10 sec
372°C35 secstep #2, 30 times
472°C10 min
54°Cpause
Phusion/RBC PCR
PCR Mix
Mix RBC (3)
Template1µl
dNTPs0.5µl
Primer Fw1µl
Primer Rv
Buffer RBC5µl
Phusion pol0.3µl
RBC0.25µl
Water40.95µl


PCR Settings
StepTemperatureTimeGo to
194°C2 min
294°C1 min
362.5°C1 min
472°C1 min 9 secstep #2, 30 times
572°C7 min
64°Cpause

The products of these three PCRs were then loaded on a 1% agarose gel.
Gel
Loading scheme
1kb ladderGC(2)HF(1)RBC(3)
\"Gel\"
As showed by the gel, only the Phusion/RBC PCR was successful.


Purifications

Then, I purified the EX-SAMsynthetase-SP sample produced today with the Phusion/RBC PCR, and the 5 R0010 PCR insert that were amplified on tuesday 02/07.
SampleTypeQuantity
RBC(3)EX-SAMsynthetase-SP103.6ng/µl
HF1R0010 insert17.5ng/µl
HF2R0010 insert19.5ng/µl
HF3R0010 insert15.9ng/µl
G1R0010 insert17.8ng/µl
G2R0010 insert16.3ng/µl


OverNight Purification

Then, Viola was so polite to prepare the O/N digestion mixes (with the usual protocol) and incubate them at 37°C. An EX-SAMsynthetase-SP sample (RBC#3 from today, 103.6ng/µl) and a linear pSB1C3 sample (G3A from 01/07, 44.8ng/µl) were restricted with XbaI and PstI (Nebuffer2).
Digestion mixes
RBC#3G3A
Template40µl50µl
XbaI2.5µl1.5µl
PstI
NEBuffer 210µl5µl
BSA10µl5µl
Water35µl0
", "tags" : "SAMsynthetase" }