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From 2013.igem.org

{ "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:

Sample	Quantity
G1 SAMsynthetase	18.8ng/µl
G2 linear pSB1C3	6.5ng/µl

After that, I performed the ligation (the plasmid had a too low concentration).

	Ctrl	0.5:1	0.5:2
Buffer	3µl
Plasmid	14.3µl
Insert	0	6µl	12µl
Ligase	1µl
Water	11.7µl	5.7µl	0µl

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

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

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(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).

Given that R0010 is 200bp long, the PCR program was the following:
Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.
As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!

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(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.", "tags" : "SAMsynthetase-Plac-pSB1C3" }