User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/06/02

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Wednesday 2nd June 2010
Today we received luxAB and luxCDE from the Edinburg team, so I performed a hot start PCR with rtth to extract them, I made two of each because I use both the primers they sent and also our primers for prefix and suffix, the samples were labeled as follows:


 * AB1: using Edinburg primers


 * AB2: using our suffix and prefix primers


 * CDE1: using Edinburg primers


 * CDE2: using Edinburg primers

here’s the mail we received from Edinburg team:

I've sent the luxCDE

>>>>> template

>>>>> and also BioBrick K216016 which is a composite BioBrick containing

>>>>> luxAB, with the PyeaR promoter; you can use this to check activity.

>>>>> It

>>>>> is induced by nitrate.

>>>>>

>>>>> I've also included forward and reverse primers for luxAB and

>>>>> luxCDE.

>>>>> The primers are all at 50 pmol/ul. The sequences are:

>>>>>

>>>>> BBluxABf1: tctagagctc aaatagcaatataaggac

>>>>> BBluxABr1: ccctactagta ttattaggtatattccatgtgg

>>>>> BBluxCf1: atcgaattcgcggccgcttctagag taatttaaggagattgtatg

>>>>> BBluxEr1: atcacatagta cacttacaattaggcaaagg

>>>>>

>>>>> You'll be able to clone luxCDE as EcoRI/SpeI, but the luxAB primers

>>>>> are designed to clone it as a SacI/SpeI fragment (I have a set of

>>>>> construction vectors with a SacI site overlapping the XbaI site);

>>>>> the

>>>>> forward primer does have an XbaI site but it is right at the end of

>>>>> the primer so may not cut effectively. You might have to clone the

>>>>> PCR

>>>>> product in a TA vector first before cutting it out, or

>>>>> alternatively

>>>>> order a new forward primer with an EcoRI site.

>>>>>

>>>>> Now that I think about it, it would have been simpler to include

>>>>> one

>>>>> of my construction vectors with the SacI site. The main one I use

>>>>> is

>>>>> J33207, which has Plac+lacZ', so you can cut out the insert,

>>>>> replace

>>>>> it with a SacI/SpeI insert, and select white colonies on Xgal/IPTG

>>>>> plates. It was in the 2008 distribution, if you still have it, but

>>>>> apparently not any of the subsequent ones.

Then I extracted and transformed by heatshock the lambda cI inverter (BBa_Q04510) from kit plate 1 (2009), well: 18B.

After I put it to incubate overnight with a negative control in petri boxes with kanamycin.


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