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Thursday 16th July

Lab Work

Plasmid extraction

by Johan

• BBa_S03518
• BBa_B0015

Cultures from the 07/15/2015 With the Nucleospin Kit from Macherey Nagel

Digestion

by Coralie

• BBa_S03518
• BBa_B0015
• BBa_I13602

In each tube:

• Plasmid: 10µL
• Enzyme: 1µL of each enzyme
• Buffer FastDigest (10x): 2µL
• H2O: 6µL

Enzymes choice:

• BBa_S03518 #1: XbaI + PstI
• BBa_S03518 #2: SpeI + EcoRI
• BBa_B0015 #1: PstI + SpeI
• BBa_B0015 #2: XbaI + EcoRI
• BBa_I13602 (x2): XbaI + Pst I

Incubation 1h30, 37°C

PCR

by Coralie

• BBa_R0051

We use the rehydrated plasmid from the iGEM plate 2014

Reaction mix for 3 tubes:

• GC Buffer (5x): 30µL
• dNTP (10mM): 3µL
• Forward Primer (1/10): 7,5µL
• Reverse Primer (1/10): 7,5µL
• Template DNA R0051 (2014): 5µL
• DNA Pol Phusion: 1,5µL
• H2O: 97,5µL

We use 50µL of that mix in each tube

Cycle: Initiation: 98°C - 30seconds Cycle (34 repeats): 98°C - 10seconds / 65°C - 30seconds / 72°C - 20seconds Term.: 72°C - 5min Keep it at 4°C

New culture

by Seong Koo

Observation of our plates: a lot of colony in each one. New liquid culture of:

• BBa_K1399005
• BBa_K1399019
• BBa_K1399023
• Ligation product: BBa_J23101 + BBa_K115017

2x 5ml LB + 10μl Chloramphenicol + 1 bacterial colony. We incubate cultures at 37°C, ON.

Plasmid extraction

by Pauline

• BBa_R0051
• BBa_B0030

Cultures from the 07/15/2015 With the Nucleospin Kit from Macherey Nagel

Digestion

by Pauline

• BBa_R0051 (07/15/2015)
• BBa_R0051 (07/16/2015)
• BBa_B0030
• BBa_J23101 + BBa_I13504
• BBa_J23106 + BBa_I13504
• BBa_J23117 + BBa_I13504

Reaction mix:

• Plasmid: 2µL
• EcoRI: 0,5µL
• PstI: 0,5µL
• Buffer FastDigest (10x): 2µL
• H2O: 15µL

Electrophoresis

by Pauline

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V

Digestion verification, from left to right: 1. DNA Ladder, 2. BBa_R0051 (15/07), 3. BBa_R0051 (16/07), 4. BBa_B0030#1, 5. BBa_J23101+I13504#1, 6. BBa_J23106+I13504#1, 7. BBa_J23117+I13504#1, 8. Empty, 9. Empty, 10. Empty

Purification of BioBricks by electrophoresis

by Coralie

• BBa_S03518
• BBa_I13602

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V

Purificatin verification, from left to right: 1. DNA Ladder, 2. BBa_S03518#1, 3. BBa_S03518#2, 4. BBa_I13602#1, 5. BBa_I13602#2, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty

We cut interested bands with a scalpel.

DNA Extraction and purification

by Coralie

• BBa_S03518 #1 and #2
• BBa_B0015 #1 and #2
• BBa_I13602 (x2)
• BBa_R0051 (from PCR)

We use the PCR Clean Up / Gel Extraction Kit from Macherey-Nagel We elute DNA in 30 µL.

Quantification on Agarose Gel

by Johan

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET We load 2µL of previous purified DNA with 2 µL of DNA Loading (6x) and 8 µL of H2O Migration 0,06A 80V

Quantification, from left to right: 1. DNA Ladder, 2. BBa_S03518#1, 3. BBa_S03518#2, 4. BBa_B0015#1, 5. BBa_B0015#2, 6. BBa_I13602#1, 7. BBa_I13602#2, 8. BBa_R0051#1, 9. BBa_BBa_R0051#2, 10. Empty, 11. Empty, 12. Empty

We can observe that the PCR of R0051 was effective. We can quantify purified DNA:

• BBa_S03518 #1: 15 ng/µL
• BBa_S03518 #2: 15ng/µL
• BBa_B0015 #1: 10 ng/µL
• BBa_B0015 #2: 10 ng/µL
• BBa_I13602 (x2): 5ng/µL
• BBa_R0051: 10 ng/µL

Members present:

• Instructors and advisors: Alice.
• Students: Johan, Seong Koo, Coralie, Pauline

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