Team:UNITN-Trento/Notebook/Labposts/07/04

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{"date" : "2013-07-02","author" : "gabriele","title" : "Another busy day","content" : "

SAM synthetase ligation in (linear) pSB1C3

First of all, early in the morning I added 1µl of DpnI to the SAMsynthetase#G2 O/N digestion and 1µl of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37°C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80°C).

Then, I purified and quantified the two digestion samples:
SampleQuantity
G1 SAMsynthetase18.8ng/µl
G2 linear pSB1C36.5ng/µl

After that, I performed the ligation (the plasmid had a too low concentration).
Ctrl0.5:10.5:2
Buffer3µl
Plasmid14.3µl
Insert06µl12µl
Ligase1µl
Water11.7µl5.7µl0µl

And, \"finally\", I transformed the ligations in NEB10β cells.


(2) SAMsynthetase amplification

I amplified SAMsynthetase from the E2 sample (that was purified yesterday) following the usual protocol. The PCR was performed in triplicates.

The PCR products were run on a 1% agarose gel.
Gel
Loading scheme
E1AE1BE1Cempty1kb ladder
\"Gel\"

As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.


(3) R0010 amplification

I also amplified R0010 from A sample (R0010, 243.7ng/µl) from 27/06. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86°C) and the suffix Rv (Tm = 90°C).
PCR Mixes
Mix HF (x3)Mix GC (x2)
Phusion GC buffer010µl
Phusion HF buffer10µl0
dNTPs1µl
primer Fw2.5µl
primer Rv2.5µl
template (50ng/µl)0.5µl
Phusion pol0.5µl
Water33µl

Given that R0010 is 200bp long, the PCR program was the following:
PCR settings
PCR setting
StepTemperatureTimeGo to
198°C30 sec
298°C10 sec
372°C3 secstep #2, 30 times
472°C10 min
54°Cpause

Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.
Gel
Loading scheme
100bp ladder
AHF1
AHF2
AHF3
AGC1
AGC2
empty
1kb ladder
\"Gel\"

As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!


(4) linear pSB1C3 amplification

I didn't know that linear pSB1C3 is \"impossible\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.
Gel
Loading scheme
1kb ladderG3AG3BG3C
\"Gel\"
","tags" : "SAMsynthetase-Plac-pSB1C3"}