User:DavidC/25 September 2009

From 2009.igem.org

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=== Friday the 25th ===
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==== Ligation ====
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 +
Ligation between BBa_P1003 and BBa_B0014 : <br>
 +
 +
1. Plasmid (P1003) = 0,5µL <br>
 +
1. Insert (B0014) = 3µL <br>
 +
 +
2. Plasmid (P1003) = 0,5µL <br>
 +
2. Insert (B0014) = 7µL <br>
 +
 +
Ligation between BBa_C0012 and BBa_B0014 : <br>
 +
 +
1. Plasmid (C0012) = 0,5µL <br>
 +
1. Insert (B0014) = 3µL <br>
 +
 +
2. Plasmid (C0012) = 0,5µL <br>
 +
2. Insert (B0014) = 7µL <br>
 +
 +
Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100 : <br>
 +
 +
1. Plasmid (B0030) = 1µL <br>
 +
1. Insert (B0014+J23100) = 4µL <br>
 +
 +
2. Plasmid (B0030) = 1µL <br>
 +
2. Insert (B0014+J23100) = 7µL <br>
 +
 +
3. Plasmid (B0030) = 0,5µL <br>
 +
3. Insert (B0014+J23100) = 10µL <br><br>
 +
 +
Ligation between BBa_R0010 and BBa_B0014 : <br>
 +
 +
1. Plasmid (R0010) = 0,5µL <br>
 +
1. Insert (B0014) = 2µL <br>
 +
 +
2. Plasmid (R0010) = 0,5µL <br>
 +
2. Insert (B0014) = 3µL <br>
 +
 +
3. Plasmid (R0010) = 0,5µL <br>
 +
3. Insert (B0014) = 1µL <br>
 +
 +
4. Plasmid (R0010) = 1µL <br>
 +
4. Insert (B0014) = 5µL <br><br>
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 +
Ligation between BBa_I712669 and BBa_B0014: <br>
 +
 +
1. Plasmid (I712669) = 1µL <br>
 +
1. Plasmid (B0014) = 5µL <br>
 +
 +
2. Plasmid (i712669) = 1µL <br>
 +
2. Plasmid (B0014) = 7µL <br>
 +
 +
==== NEB Enzymes ====
 +
 +
For each samples add sterilized water to obtain a maximum volume equals to 8µL. <br>
 +
 +
Ligation mix (NEB): <br>
 +
7,5µL of T4 ligase + 15µL of T4 ligase buffer. <br>
 +
Add 1,5µL / tube. <br>
 +
Incubation 1h at RT. <br>
 +
 +
==== Electroporation ====
 +
 +
Electroporation cuvettes = 2mm ; inoculums of electrocompetent <i>E.coli</i> DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation. <br>
 +
 +
Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL). <br>

Latest revision as of 03:42, 21 October 2009

framless



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October
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Contents

Friday the 25th

Ligation

Ligation between BBa_P1003 and BBa_B0014 :

1. Plasmid (P1003) = 0,5µL
1. Insert (B0014) = 3µL

2. Plasmid (P1003) = 0,5µL
2. Insert (B0014) = 7µL

Ligation between BBa_C0012 and BBa_B0014 :

1. Plasmid (C0012) = 0,5µL
1. Insert (B0014) = 3µL

2. Plasmid (C0012) = 0,5µL
2. Insert (B0014) = 7µL

Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100 :

1. Plasmid (B0030) = 1µL
1. Insert (B0014+J23100) = 4µL

2. Plasmid (B0030) = 1µL
2. Insert (B0014+J23100) = 7µL

3. Plasmid (B0030) = 0,5µL
3. Insert (B0014+J23100) = 10µL

Ligation between BBa_R0010 and BBa_B0014 :

1. Plasmid (R0010) = 0,5µL
1. Insert (B0014) = 2µL

2. Plasmid (R0010) = 0,5µL
2. Insert (B0014) = 3µL

3. Plasmid (R0010) = 0,5µL
3. Insert (B0014) = 1µL

4. Plasmid (R0010) = 1µL
4. Insert (B0014) = 5µL

Ligation between BBa_I712669 and BBa_B0014:

1. Plasmid (I712669) = 1µL
1. Plasmid (B0014) = 5µL

2. Plasmid (i712669) = 1µL
2. Plasmid (B0014) = 7µL

NEB Enzymes

For each samples add sterilized water to obtain a maximum volume equals to 8µL.

Ligation mix (NEB):
7,5µL of T4 ligase + 15µL of T4 ligase buffer.
Add 1,5µL / tube.
Incubation 1h at RT.

Electroporation

Electroporation cuvettes = 2mm ; inoculums of electrocompetent E.coli DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation.

Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL).

Retrieved from "http://2009.igem.org/User:DavidC/25_September_2009"