Team:UNITN-Trento/Notebook/Labposts/07/44
From 2013.igem.org
{ "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
Then I digested 800 ng of each sample with EcoR1 HF and Pst1 following the digestion protocol with the following results: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 |
Gel order
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 .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 |
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" }