Team:IPN-UNAM-Mexico/Notebook/August

Number	Biobrick	Restriction
2	BBa_R0079	S & P
3	BBa_F1610	X & P
4	BBa_K091146	S & P
5	BBa_K093005	X & P
6	BBa_K081016	X & P
7	BBa_EC840	X & P
8	BBa_K081009	X & P
9	BBa_R0051	S &P
10	BBa_J06800	X & P
11	BBa_Q04121	X & P
12	BBa_B0034	S & P
13	BBa_C0079	E & S
14	BBa_C0179	E & S
15	BBa_B0015	E & X
16	BBa_K081018	E & S
17	BBa_K116640	X & P
23	BBa_S03154	X & P
24	BBa_k145201	E & S

06-Agust-2009

Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.
We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.

07-Agust-2009

Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.

10-Agust-2009

We made DNA purification from the Gel an we are planing start ligations this week.

11-Agust-2009

Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations.

12-Agust-2009

We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).

In this step we use 2 as backbone and 3 as insert, on the same 15 as backbone and 13 as insert, for 2-3 (BBa_K266000) we expect 3034 bp band as shown below, for 15-13 4074 bp. 4 as backbone 23 as insert. For ligation we use purifed DNA from agarosa Gel.

Mix Ligation	Per reaction
Plasmidic DNA	5μl
Insert DNA	25μl
T4 DNA Ligasa Buffer	3μl
T4 DNA ligasa	2μl
ATP	3μl

The ligation left 17 hours overnight in 14ºC.

13-Agust-2009

We transform the ligation by thermic shock and plate. We received primers for 11 and 12.

14-Agust-2009

The transformation was successful we get colonies, we picket out for plasmid and left in 37ºC overnight.

15-Agust-2009

Only take off culture from camera and prepare for monday mini prep.

17-Agust-2009

We did mini prep and run out on a gel, we expect 3034 bp for L2_3(BBa_K266000) and 4074 bp for L15_13 (Bba_K266002) this are successful not so for 4_23 this is unsuccessful and proceed to repeat the ligation.

"Check plasmid and gel picutre below"

18-Agust-2009

The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight)

19-Agust-2009

Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.

20-Agust-20009

Purificated from gel for begin ligation.

21-Agust-2009

The transformation was through thermick shok and plate the weight we expect is amplicon 1515 bp 15_13 4074 bp total for Bba_K266003 5574 bp.

"Check plasmid and gel picutre below"

22-Agust-2009

The transformation was successful and we get colonies picket out for midi prep. For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.

23-Agust-2009

Meeting; we are detected a mistake on the primer, our primer musted include J23100 promotor now we have to do a extra ligation, and use J23100 as backbone.

24-Agust-2009

We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight

25-Agust-2009

Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation.

"Check plasmid and gel picutre below"

For ligation we going to do a mix and leave 17 hours in 14ºC camera.

26-Agust-2009

We transform by thermic shock and plate.

27-Agust-2009

The transformation was unsuccessful we haven't colonies. We going to repeat the ligation and left overnight incubation 14ºC.

@@ Line 280: / Line 280: @@
 "Check plasmid and gel picutre below"
-For ligation we going to do a mix and leave 17 hours in 14º camera.
+For ligation we going to do a mix and leave 17 hours in 14ºC camera.
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