Team:UNITN-Trento/Notebook/Labposts/07/44

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Revision as of 07:51, 9 September 2013

{ "date" : "2013-07-05", "author" : "emil", "title" : " Purification of the second run of Inocula of ligation and re-ligation of the digested products ", "content" : " I added 1µl of DPN1 to the insert and 1µl of SAP to the backbones.Then I have purified the inocula following the miniprep protocol.Afterwards I quantified them toghether with the results of the purification of the digestion products(performed following the purification protocol ) with the following results:

Quantification

Sample	Quantification
1:1 024+0840 E	482.6ng/µl
1:1 024+0840 E	700ng/µl
1:1 024+0840	373.1ng/µl
1:3 024+0840	423.5ng/µl
1:3 024+0840	327.8ng/µl
1:1 026+0840	293.1ng/µl
1:1 026+0840	1243ng/µl
1:3 026+0840	766.4ng/µl
1:3 026+0840	369.1ng/µl
GFP	15.9ng/µl
K823026	36.3ng/µl
K823026	43.7ng/µl

Then I digested 800 ng of each sample with EcoR1 HF and Pst1 following the digestion protocol with the following results:

Gel order

Sample	Well
Ladder 1kb Fermentas	1
BBa_K823024+BBa_E0840 A	3
BBa_K823024+BBa_E0840 B	5
BBa_K823024+BBa_E0840 C	6
BBa_K823024+BBa_E0840 D	7
BBa_K823024+BBa_E0840 E	8
BBa_K823026+BBa_E0840 F	9
BBa_K823026+BBa_E0840 G	10
BBa_K823026+BBa_E0840 H	11
BBa_K823026+BBa_E0840 I	12

Gel image

As we can see no one of the wells shows the right insert in the lower band(there is always the previous insert the RFP + Lac promoter ~ 1200 bp).At the same time I did the ligation of K823024+E0840 and K823026+E0840 previously digested following the ligation protocol .
N.B. Remember always to spin the ligation buffer before using
After that I plated the product of the ligation as done before(1:1,1:3,ctrl for each sample) on Ampicillin added LB.", "tags" : "K832024-K823026-E0840" }