User:DavidC/24 September 2009

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=== Thursday the 24th ===
 +
 +
==== Restriction digest ====
 +
 +
==== Ligation between BBa_P1003 and BBa_B0014 ====
 +
 +
Restriction digest of BBa_P1003 (3675bp) by Spe and Pst: <br>
 +
 +
DNA (10µg final) = 4µL <br>
 +
Buffer H (TAKARA) =2µL <br>
 +
H20 = 12µL <br>
 +
Spe1 (TAKARA) = 1µL <br>
 +
Pst1 (TAKARA) = 1µL <br>>
 +
Incubation 1h at 37°C. <br>
 +
 +
Restriction digest of BBa_B0014 (95bp) by XbaI and PstI: <br>
 +
 +
DNA (10µg final) = 4µL <br>
 +
Buffer M (TAKARA) =2µL <br>
 +
H20 = 12µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
Pst1 (TAKARA) = 1µL <br>>
 +
Incubation 1h at 37°C. <br>
 +
 +
==== Ligation between BBa_C0012 and BBa_B0014 ====
 +
 +
Restriction digest of BBa_C0012 (3207bp) by PstI and SpeI: <br>
 +
 +
DNA (10µg final) = 6,47µL <br>
 +
Buffer H (TAKARA) =2µL <br>
 +
H20 = 9,63µL <br>
 +
Spe1 (TAKARA) = 1µL <br>
 +
Pst1 (TAKARA) = 1µL <br>>
 +
Incubation 1h at 37°C. <br>
 +
 +
Restriction digest of BBa_BB014 (95bp) by XbaI and PstI: <br>
 +
 +
DNA (10µg final) = 4,16µL <br>
 +
Buffer H (TAKARA) =2µL <br>
 +
H20 = 11,84µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
Pst1 (TAKARA) = 1µL <br>>
 +
Incubation 1h at 37°C. <br>
 +
 +
==== Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100 ====
 +
 +
Restriction digest of BBa_B0030 (2094bp) by XbaI and EcoRI: <br>
 +
 +
DNA (10µg final) = 10,25µL <br>
 +
Buffer M (TAKARA) =2µL <br>
 +
H20 = 2,75µL <br>
 +
EcoR1 (TAKARA) = 1µL <br>
 +
Xba1 (TAKARA) = 1µL <br>>
 +
Incubation 1h at 37°C. <br>
 +
 +
Restriction digest of BBa_J23100 + BBa_B0014  by SpeI and EcoRI: <br>
 +
 +
DNA (10µg final) = 2,57µL <br>
 +
Buffer M (TAKARA) =2µL <br>
 +
H20 = 13,43µL <br>
 +
EcoR1 (TAKARA) = 1µL <br>
 +
Spe1 (TAKARA) = 1µL <br>>
 +
Incubation 1h at 37°C. <br>
 +
 +
==== Ligation between BBa_R0010 and BBa_B0014 ====
 +
 +
Restriction digest of BBa_R0010 (2279bp) by XbaI and EcoRI: <br>
 +
 +
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 ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
 +
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 ====
 +
 +
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>

Latest revision as of 15:59, 21 October 2009

framless



August
MTWTFSS
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10 11 12 13 14 15 16
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24 25 26 27 28 29 30
31
September
MTWTFSS
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14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31


Contents

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.

Retrieved from "http://2009.igem.org/User:DavidC/24_September_2009"