Difference between revisions of "Team:Paris Saclay/Notebook/July/10"
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| + | {{Team:Paris_Saclay/notebook_header}} | ||
=Friday 10th July= | =Friday 10th July= | ||
==Lab Work== | ==Lab Work== | ||
| − | + | ===PCR=== | |
| − | + | ||
''by Coralie'' | ''by Coralie'' | ||
| Line 22: | Line 22: | ||
Keep it at 4°C | Keep it at 4°C | ||
| − | + | ===Verification of PCR products by electrophoresis=== | |
''by Coralie'' | ''by Coralie'' | ||
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET | Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET | ||
Migration 0,06A 80V | Migration 0,06A 80V | ||
| + | |||
| + | [[File:ParisSaclay_10.07.15_-_PCR_tige_boucle.jpg|300px|center]] | ||
| + | <html><i><p>Verification of PCR products, from left to right: 1. Empty, 2. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017, 6. Empty</i></p></i></html> | ||
We confirm that the PCR was effective. We can continue the protocol. | We confirm that the PCR was effective. We can continue the protocol. | ||
| − | + | ===Digestion=== | |
''by Coralie'' | ''by Coralie'' | ||
| Line 43: | Line 46: | ||
Incubation at 37°C for 1 hour | Incubation at 37°C for 1 hour | ||
| − | + | ===Purification of the digested PCR product=== | |
''by Coralie'' | ''by Coralie'' | ||
| Line 49: | Line 52: | ||
Keep the product at -20°C | Keep the product at -20°C | ||
| − | + | ===Quantification of the PCR product=== | |
''by Coralie'' | ''by Coralie'' | ||
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET | Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET | ||
Migration 0,06A 80V | Migration 0,06A 80V | ||
| + | |||
| + | [[File:ParisSaclay_10.07.15_ARN_tige_boucle_dig_purif.jpg|300px|center]] | ||
| + | <html><i><p>Quantification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K115017, 3. BBa_K115017, 4. Empty, 5. Empty, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty</i></p></i></html> | ||
Concentration of the digested and purified PCR product: 50ng/µL | Concentration of the digested and purified PCR product: 50ng/µL | ||
| − | + | ===Transformation=== | |
''by Johan, Seong Koo'' | ''by Johan, Seong Koo'' | ||
| − | * | + | * BBa_S03518 |
| − | * | + | * BBa_B0030 |
| − | * | + | * BBa_B0015 |
| − | * | + | * BBa_K1399005 |
| − | + | ===New culture=== | |
''by Johan'' | ''by Johan'' | ||
New liquid culture of: | New liquid culture of: | ||
| − | * | + | * BBa_R0051 (2015) |
5ml LB + 10μl Ampicilline + 1 bacterial colony. | 5ml LB + 10μl Ampicilline + 1 bacterial colony. | ||
We incubate cultures at Room temperature for 4days. | We incubate cultures at Room temperature for 4days. | ||
| − | + | ==Human Practices== | |
| − | ==Meeting with Jacques Livage== | + | ===Meeting with Jacques Livage=== |
| − | + | ||
'''Members present:''' | '''Members present:''' | ||
*Instructors : Alice. | *Instructors : Alice. | ||
*Students : Johan, Pauline, Coralie, Audrey, Seong Koo. | *Students : Johan, Pauline, Coralie, Audrey, Seong Koo. | ||
| + | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 21:15, 18 September 2015
Contents
Friday 10th July
Lab Work
PCR
by Coralie
PCR Mix (for 3 tubes)
- GC Buffer: 30µL
- dNTP 10mM: 3µL
- Forward Primer (dilution: 1/10e): 7,5µL
- Reverse Primer (dilution: 1/10): 7,5µL
- Template DNA K115017 (dilution 1/10e): 6µL
- DNA polymerase Phusion: 1,5µL
- H2O: 94,5µL
In each tube: 50µL from the mix
Cycle: Initiation: 98°C - 30seconds Cycle (30 repeats): 98°C - 10seconds / 53°C - 30seconds / 72°C - 10seconds Term.: 72°C - 5min Keep it at 4°C
Verification of PCR products by electrophoresis
by Coralie
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V
Verification of PCR products, from left to right: 1. Empty, 2. DNA Ladder, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017, 6. Empty
We confirm that the PCR was effective. We can continue the protocol.
Digestion
by Coralie
We digest 2 tubes of the PCR product Mix for each tube:
- XbaI: 1µL
- PstI: 1µL
- FastDigest Buffer: 2µL
- H2O: µL
- PCR product: 10µL
Incubation at 37°C for 1 hour
Purification of the digested PCR product
by Coralie
We use the Nucleospin kit from Magerey Nagel Keep the product at -20°C
Quantification of the PCR product
by Coralie
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V
Quantification, from left to right: 1. DNA Ladder, 2. BBa_K115017, 3. BBa_K115017, 4. Empty, 5. Empty, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty, 11. Empty
Concentration of the digested and purified PCR product: 50ng/µL
Transformation
by Johan, Seong Koo
- BBa_S03518
- BBa_B0030
- BBa_B0015
- BBa_K1399005
New culture
by Johan
New liquid culture of:
- BBa_R0051 (2015)
5ml LB + 10μl Ampicilline + 1 bacterial colony. We incubate cultures at Room temperature for 4days.
Human Practices
Meeting with Jacques Livage
Members present:
- Instructors : Alice.
- Students : Johan, Pauline, Coralie, Audrey, Seong Koo.
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France
Copyright © 2015 iGEM Paris Saclay. *This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.