Team:UNITN-Trento/Notebook/Labposts/07/01

From 2013.igem.org

(Difference between revisions)

Revision as of 09:39, 3 October 2013

{ "date" : "2013-07-01", "author" : "emil", "title" : " Re-screening, amplification and digestion(together with the two B. subtilis backbones) of the GFPs E0840 and E0240 ", "content" : "I screened the three other sample of GFP(0840/2,0840/4,0240/1,0240/3) like in the\"> previous post with the following results:(the gel was run together with Girelli's sample, we are interested in the first 4 sample)

Gel order

Sample	Well
BBa_E0840/2	2
BBa_E0840/4	3
BBa_E0240/1	4
BBa_E0240/3	5
Ladder 1kb Fermentas	1

Gel image

We can see 2 bands for each sample: the upper is the plasmid(pSB1A2 2079 bp), the lower is presumably the GFP(BBa_E0240 826 bp,BBa_E0840 828 bp)(prefix and suffix escluded)at 900 bp.Afterwards i have amplified 0.5 6micro;l of one of the 4 sample(E0840/4 475.9 ng/micro;l) following the PCR protocol and exploiting the universal primers(prefix Forward,Suffix Reverse) with the following resuts(as usual I did a gel to verify the pcr):

Gel order

Sample	Well
BBa_E0840/4 a	2
BBa_E0840/4 b	3
BBa_E0840/4 c	4
Ladder 1kb Fermentas	1

Gel image

As we can see the pcr completed succesfully, there are the right bands at 900-1000 bp.Then I purified the pcr following the purification protocol . After that I have quantified the product with the following results:

Quantification

Sample	Quantification
BBa_E0840/4 a	30.5ng/µl
BBa_E0840/4 b	44ng/µl
BBa_E0840/4 c	42.6ng/µl

Finally I have digested o/n all the 48.5µl of b sample and of 2 sample of the backbone(K823026 359.7ng/µl,K823024 334.4ng/µl) with EcoR1-HF and Pst1 following the digestion protocol. ", "tags" : "K832024-K823026-E0840-E0240" }