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Revision as of 15:34, 21 October 2009 (view source) DavidC (Talk | contribs) (→Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100) ← Older edit		Latest revision as of 15:59, 21 October 2009 (view source) DavidC (Talk | contribs) (→DNA electrophoresis)
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Line 69:		Line 69:
	Incubation 1h at 37°C. <br>		Incubation 1h at 37°C. <br>
-	Restriction digest of BBa_B0014 + BBa_J23100 by SpeI and EcoRI: <br>	+	Restriction digest of BBa_J23100 + BBa_B0014  by SpeI and EcoRI: <br>
	DNA (10µg final) = 2,57µL <br>		DNA (10µg final) = 2,57µL <br>
Line 80:		Line 80:
	==== Ligation between BBa_R0010 and BBa_B0014 ====		==== Ligation between BBa_R0010 and BBa_B0014 ====
		+	Restriction digest of BBa_R0010 (2279bp) by XbaI and EcoRI: <br>
-	==== Ligation between BBa_I712612 and BBa_B0014 ====	+	DNA (10µg final) = 55,6µL <br>
		+	Buffer H (TAKARA) =6µL <br>
		+	H20 = 5,4µL <br>
		+	EcoR1 (TAKARA) = 1µL <br>
		+	Xba1 (TAKARA) = 1µL <br>>
		+	Incubation 1h at 37°C. <br>
		+	Restriction digest of BBa_B0014 (95bp) by SpeI and EcoRI: <br>
		+
		+	DNA (10µg final) = 4,16µL <br>
		+	Buffer H (TAKARA) =2µL <br>
		+	H20 = 11,84µL <br>
		+	EcoR1 (TAKARA) = 1µL <br>
		+	Spe 1 (TAKARA) = 1µL <br>>
		+	Incubation 1h at 37°C. <br>
		+
		+	==== Ligation between BBa_I712669 and BBa_B0014 ====
		+
		+	Restriction of BBa_I712669 (4806bp) by SpeI and PstI: <br>
		+
		+	DNA (10µg final) = 4µL <br>
		+	Buffer H (TAKARA) =2µL <br>
		+	H20 = 12µL <br>
		+	Pst1 (TAKARA) = 1µL <br>
		+	Spe 1 (TAKARA) = 1µL <br>>
		+	Incubation 1h at 37°C. <br><br>
		+
		+	Restriction digest of B0014 (95bp) by XbaI and PstI: <br>
		+
		+	Same sample as the ligation between BBa_C0012 and BBa_B0014. <br>
	==== DNA electrophoresis ====		==== DNA electrophoresis ====
Line 91:		Line 120:
-	Samples:	+	Samples: BBa_C0012, BBa_BB014 (XbaI, PstI), BBa_B0030, BBa_J23100 + BBa_B0014, BBa_R0010, BBa_B0014 (SpeI, EcoRI), BBa_I712669. <br>
		+
		+	We have not the picture (technical problem). <br>
	==== DNA purification ====		==== DNA purification ====
	Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>		Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>

---

Latest revision as of 15:59, 21 October 2009

August M T W T F S S           [http://2009.igem.org/User:DavidC/1_August_2009 1] [http://2009.igem.org/User:DavidC/2_August_2009 2] [http://2009.igem.org/User:DavidC/3_August_2009 3] [http://2009.igem.org/User:DavidC/4_August_2009 4] [http://2009.igem.org/User:DavidC/5_August_2009 5] [http://2009.igem.org/User:DavidC/6_August_2009 6] [http://2009.igem.org/User:DavidC/7_August_2009 7] [http://2009.igem.org/User:DavidC/8_August_2009 8] [http://2009.igem.org/User:DavidC/9_August_2009 9] [http://2009.igem.org/User:DavidC/10_August_2009 10] [http://2009.igem.org/User:DavidC/11_August_2009 11] [http://2009.igem.org/User:DavidC/12_August_2009 12] [http://2009.igem.org/User:DavidC/13_August_2009 13] [http://2009.igem.org/User:DavidC/14_August_2009 14] [http://2009.igem.org/User:DavidC/15_August_2009 15] [http://2009.igem.org/User:DavidC/16_August_2009 16] [http://2009.igem.org/User:DavidC/17_August_2009 17] [http://2009.igem.org/User:DavidC/18_August_2009 18] [http://2009.igem.org/User:DavidC/19_August_2009 19] [http://2009.igem.org/User:DavidC/20_August_2009 20] [http://2009.igem.org/User:DavidC/21_August_2009 21] [http://2009.igem.org/User:DavidC/22_August_2009 22] [http://2009.igem.org/User:DavidC/23_August_2009 23] [http://2009.igem.org/User:DavidC/24_August_2009 24] [http://2009.igem.org/User:DavidC/25_August_2009 25] [http://2009.igem.org/User:DavidC/26_August_2009 26] [http://2009.igem.org/User:DavidC/27_August_2009 27] [http://2009.igem.org/User:DavidC/28_August_2009 28] [http://2009.igem.org/User:DavidC/29_August_2009 29] [http://2009.igem.org/User:DavidC/30_August_2009 30] [http://2009.igem.org/User:DavidC/31_August_2009 31]

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Contents 1 Thursday the 24th 1.1 Restriction digest 1.2 Ligation between BBa_P1003 and BBa_B0014 1.3 Ligation between BBa_C0012 and BBa_B0014 1.4 Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100 1.5 Ligation between BBa_R0010 and BBa_B0014 1.6 Ligation between BBa_I712669 and BBa_B0014 1.7 DNA electrophoresis 1.8 DNA purification

Thursday the 24th

Restriction digest

Ligation between BBa_P1003 and BBa_B0014

Restriction digest of BBa_P1003 (3675bp) by Spe and Pst:

DNA (10µg final) = 4µL
Buffer H (TAKARA) =2µL
H20 = 12µL
Spe1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Restriction digest of BBa_B0014 (95bp) by XbaI and PstI:

DNA (10µg final) = 4µL
Buffer M (TAKARA) =2µL
H20 = 12µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Ligation between BBa_C0012 and BBa_B0014

Restriction digest of BBa_C0012 (3207bp) by PstI and SpeI:

DNA (10µg final) = 6,47µL
Buffer H (TAKARA) =2µL
H20 = 9,63µL
Spe1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Restriction digest of BBa_BB014 (95bp) by XbaI and PstI:

DNA (10µg final) = 4,16µL
Buffer H (TAKARA) =2µL
H20 = 11,84µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100

Restriction digest of BBa_B0030 (2094bp) by XbaI and EcoRI:

DNA (10µg final) = 10,25µL
Buffer M (TAKARA) =2µL
H20 = 2,75µL
EcoR1 (TAKARA) = 1µL
Xba1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Restriction digest of BBa_J23100 + BBa_B0014 by SpeI and EcoRI:

DNA (10µg final) = 2,57µL
Buffer M (TAKARA) =2µL
H20 = 13,43µL
EcoR1 (TAKARA) = 1µL
Spe1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Ligation between BBa_R0010 and BBa_B0014

Restriction digest of BBa_R0010 (2279bp) by XbaI and EcoRI:

DNA (10µg final) = 55,6µL
Buffer H (TAKARA) =6µL
H20 = 5,4µL
EcoR1 (TAKARA) = 1µL
Xba1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Restriction digest of BBa_B0014 (95bp) by SpeI and EcoRI:

DNA (10µg final) = 4,16µL
Buffer H (TAKARA) =2µL
H20 = 11,84µL
EcoR1 (TAKARA) = 1µL
Spe 1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Ligation between BBa_I712669 and BBa_B0014

Restriction of BBa_I712669 (4806bp) by SpeI and PstI:

DNA (10µg final) = 4µL
Buffer H (TAKARA) =2µL
H20 = 12µL
Pst1 (TAKARA) = 1µL
Spe 1 (TAKARA) = 1µL
> Incubation 1h at 37°C.

Restriction digest of B0014 (95bp) by XbaI and PstI:

Same sample as the ligation between BBa_C0012 and BBa_B0014.

DNA electrophoresis

85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.

Samples: BBa_C0012, BBa_BB014 (XbaI, PstI), BBa_B0030, BBa_J23100 + BBa_B0014, BBa_R0010, BBa_B0014 (SpeI, EcoRI), BBa_I712669.

We have not the picture (technical problem).

DNA purification

Kit Qiagen “gel extraction kit”, final volume = 50µL.

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