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{ "date" : "2013-06-14", "author" : "gabriele-emil", "title" : "What happend to SAMsynthetase ligation?", "content" : "Got the inocula from yesterday:

  • 4 out of 5 inocula of SAMsynthetase+pSB1C3[no_RFP] were good, one (3) didn't grow.
  • 4 out of 5 inocula of SAMsynthetase+pSB1C3[RFP] were good, one (5R) was red!

Then, we miniprepped the samples (except for the red, 5R, one) using the protocol. We chose to miniprep also the inocula that did not grow (3), just as a control. After miniprep, quantification was performed with nanodrop.
Quantification results
SampleDNA [ng/µl]SampleDNA [ng/µl]

After that, we digested an aliquote of 800ng of each DNA sample with EcoRI and PstI using our digestion protocol, putting the digestion mix to incubate for 1.5h at 37°C (static).
Digestion mix
Buffer 10X (Nebuffer4)2µl
BSA 10X1µl
Water (up to 20µl)10.4µl10.7µl10.7µl7.25µl9.9µl6.8µl9.6µl9.4µl
Total volume20µl

Then we realized that LacI+RFP and SAMsynthetase have the same base length, and that a gel would not be able to discriminate between them. So, we added 1µl of BamHI to each sample, knowing that this enzyme is able to cut SAMsynthetase (in a band of 100bp and one of 1055bp) to identify the presence of SAM.

So, we prepared a 1.5% agarose gel and performed an electrophoresis for 30 minutes at 120V.
Gel Image
Loaded samples
1kb ladder#1#2#4#5100bp ladder#1R#2R#3R#4R
From the gel is clear that the samples without RFP (1, 2, 4, 5) do not have any insert. The RFP samples (1R, 2R, 3R, 4R) might contain the SAMsynthetase, but the insert might be LacI+RFP.

To determine whether the insert was LacI+RFP or SAMsynthetase we wanted to perform a RBC Taq PCR against SAMsynthetase (SAMsynthetase-Fw and SAMsynthetase-Rv primers), but we set a wrong PCR program... next time verify the PCR program more accurately!!! On Monday we will start again from the PCR... so sad D:
", "tags" : "SAMsynthetase" }