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<div class="container"><article class="col-md-11"><h1>Laboratory Notebook</h1> | <div class="container"><article class="col-md-11"><h1>Laboratory Notebook</h1> | ||
| − | <p class="introduction-text">A laboratory notebook is our real time record of our expriments. It porvides a detailled record of exactly what we do in the laboratory to obtain our experimental results. It documents our hypotheses, experiments and initial analysis or interpretation of our experiments | + | <p class="introduction-text">A laboratory notebook is our real time record of our expriments. It porvides a detailled record of exactly what we do in the laboratory to obtain our experimental results. It documents our hypotheses, experiments and initial analysis or interpretation of our experiments. Moreover, we also mentionned the name of exprimenters and the place of our experiments. The protocols that we follow are available on protocols page: <a class="btn-sm" href="https://2018.igem.org/Team:Sorbonne_U_Paris/Protocols" role="button" style=" color:white; border-radius: 10px; background-color: #22D66C; text-decoration:none;"><i class="fas fa-book fa-1x"></i> Protocols </a> <br> |
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| − | + | </p> </article></div> | |
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<div class="d-flex justify-content-end"> <div class="chip-icone"> <div class="chip"> | <div class="d-flex justify-content-end"> <div class="chip-icone"> <div class="chip"> | ||
<img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96"> | <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96"> | ||
| − | Sara | + | Sara and Parisa |
</div> | </div> | ||
</div> </div> | </div> </div> | ||
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<div class="d-flex justify-content-end"> <div class="chip-icone"> <div class="chip"> | <div class="d-flex justify-content-end"> <div class="chip-icone"> <div class="chip"> | ||
<img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96"> | <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96"> | ||
| − | Sara | + | Sara and Parisa |
</div> | </div> | ||
<div class="chip"> | <div class="chip"> | ||
A laboratory notebook is our real time record of our expriments. It porvides a detailled record of exactly what we do in the laboratory to obtain our experimental results. It documents our hypotheses, experiments and initial analysis or interpretation of our experiments. Moreover, we also mentionned the name of exprimenters and the place of our experiments. The protocols that we follow are available on protocols page: Protocols
| June | ||||||
|---|---|---|---|---|---|---|
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | ||||
| 4 | 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 | 1 |
| July | ||||||
|---|---|---|---|---|---|---|
| M | T | W | T | F | S | S |
| 2 | 3 | 4 | 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 | |||||
| August | ||||||
|---|---|---|---|---|---|---|
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | 4 | 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 | ||
| September | ||||||
|---|---|---|---|---|---|---|
| M | T | W | T | F | S | S |
| 1 | 2 | |||||
| 3 | 4 | 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 |
| October | ||||||
|---|---|---|---|---|---|---|
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | 4 | 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 | |||||
Aurélie
Jimmy
| Quantity in µL | Mix*8 | |
|---|---|---|
| Plasmid DNA | 1 | |
| Phusion enzyme | 0.25 | 2 |
| HF buffer (5X) | 5 | 40 |
| dNTPs (10µM) | 0.5 | 4 |
| Fw Primer (10µM) | 1.25 | |
| Rv Primer (10µM) | 1.25 | |
| H20 | 15.75 | 126 |
21.5µL of mix by tube + 1µL of DNA + 2*1.25µL of primer.
pSAD promoter : primers Ig3/Ig4, template p0-07 : Expected length : 807 pb
Paromomycin CDS : primers Ig11/Ig12, template p0-12: Expected length : 1652 pb
Rubisco intron : primers Ig5/Ig6, template p0-02: Expected length : 177 pb
Cycle : 98°C, 35*(98°C - 1min, 50°C - 2min, 72°C - 3min), 72°C - 3min, 12°C - hold
Aurélie
Soukeyna
+ 5µL of loading buffer 6X in PCR product
15 µL/well, 1% agarose gel
Migration : 100V, 20 min
| Position | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|---|---|---|---|---|---|---|---|---|---|
| Content | Ladder | puc19 Ig5/Ig6 rubisco |
p0-2 Ig5/Ig6 rubisco |
puc19 Ig3/Ig4 pSAD |
p0-7 Ig3/Ig4 pSAD |
puc19 Ig11/Ig12 paromycine R |
p0-12 Ig11/Ig12 paromycine R |
H2O Ig5/Ig6 |
H2O Ig11/Ig12 |
| Expected lenght (pb) | 0 | 177 | 0 | 807 | 0 | 1 652 | 0 | 0 |
Figure 1 : Gel electrophoresis of PCR products rubisco (2&3) intron,pSAD (4&5), paromomycin(6&7)
| Fragment | Notation | Measured concentration (ng/µL) |
|---|---|---|
| Rubisco intron | i0- IGEM1 | 26,9 |
| pSAD | i0-IGEM3 | 19,3 |
| paromycin | i0-ApHVIII | 7,8 |
Note : We should re-do the PCR of “ paromycine”
- p0universal = pAGM9121
- p0B3 pICH41258
Victor
Aurélie
Soukeyna
Aim : Extraction of plasmides from yesterday’s cultures and reactions of digestion/ligation p0universal/ pSAD and p0-B3/intron rubisco.
(p0- B3 = p0B3- pICH41258)
Purification of p0universel and p0- B3 plasmides (kit used: Nucleopsin Plasmid) : Elution volume= 40µL
Measure of DNA extracts concentration using Nanodrop (2 extracts per plasmid)
- p0-universel= 71,5 ng/µl , 64,9ng/µl
- p0-B3= 92,8ng/µl, 90,7ng/µL
Mix DNA
| Tube number | 2 | 6 | 1 | 5 |
|---|---|---|---|---|
| µl plasmid | 0,68 | 0,68 | 0,97 | 0,97 |
| µl insert | 2,733 | 1,98 | ||
| µl H2O | 10,56 | 13,32 | 11,06 | 13,07 |
| pSAD | pSAD | Rubisco | Rubisco |
2: plasmide uni+ pSAD+ enzymes
6: plasmide uni + enzymes
1: plasmides B3+ introns enzymes
5: plasmides B3+ enzymes
Mid enzyme (6µl/tube) number of ligations : 4.5
| Reaction mix | µL | master mix |
|---|---|---|
| BS/HF (µl) | 1 | 4,5 |
| T4 ligase(µl) | 1 | 4,5 |
| Buffer NEB CS (µl) | 2 | 9 |
| ATP 10mM(µl) | 2 | 9 |
Cycle:
- 37°C- 10min * 3
- 16°C- 10min *3
- 37°C- 10min
- 65°C- 20min
- 16°C- ∞
Aurélie
Ursula
| Product | Quantity (µL) | Quantity (µL) |
|---|---|---|
| Plasmide de p0-12 | 1 | ↗ |
| Enzyme Phusion | 0.25 | 2 |
| HF buffer (5X) | 5 | 40 |
| dNTPs (10mM) | 0.5 | 4 |
| Primer F Ig11 (10µM) | 1.25 | 10 |
| Primer F Ig12 (10µM) | 1.25 | 10 |
| DNA | 0 | 1 |
| H20 | 15.75 | 126 |
| Total quantity | 25 | 25 |
| DNA (template) | Hybridation temperature | |
|---|---|---|
| Tube 1 | p⦽-12 | 50°C |
| Tube 2 | p⦽-12 | 52°C |
| Tube 3 | p⦽-12 | 53°C |
| Tube 4 | p⦽-12 | 54°C |
| Tube 5 | p⦽-12 | 55°C |
| Tube 6 | p⦽-12 | 157°C |
| Controle E | p⦽-19 | 50°C |
Cycle:
98°C
98°C - 1min *30
50-57°C- 2 min *30
72°C - 3 min *30
72°C - 3 min
12°C - ∞
- Addition of 5µl loading buffer 6* (5*µL in 25µl)
- Deposit of 30µl of extract on 1% agarose gel
- Migration at 90V for 40 min
Missing: PCR photo
- PCR ok with paromycine at 800pb different from the expected 1657 pb expected before
- Insert Paro stocked at -20°C
Kit used: Gene JET Plasmid-MiniprepKit- Thermo Scientific
Centrifugation 13000 RPM
Digestion to check the size of the inserts (using BsaI)
| Plasmid(µl) | 5 | 5 |
|---|---|---|
| Cut Smart Buffer (10X) (µl) | 11 | 9C |
| Enzyme Bsa (µl) | 0.5 | 4.5 |
| Water (µl) | 3.5 | 31.5 |
Addition of 2µl of loading buffer 6* (2µl in 10µl)
Deposit of 12µl on 1% agarose gel
Migration at 90V, for 40min
Figure : Gel electrophoresis : Purification of “p⦽-IGEM1” and “p⦽-IGEM3” plasmids
We keep : 1a= rubisco intron= p⦽-IGEM1(1b too is conserved)
2a= pSAD- p⦽-IGEM3
Stock in glycerol￫ -30°C (DH5𝛂)mycologue
Note: Transplanting of the microalguae strain that was previously blocked by FedEX
Aurélie
Ursula
Launch of 2* 3.5 mL of bacterial culture → +spectromycine of pICH4 1264 which contains the destination plasmid of the “paromicycn fragment)
Aurélie
Ursula
| Culture 1 | 26,7ng/µl | 1.92 | 1.73 |
| Culture 2 | 25,7ng/µl | 1.92 | 2.02 |
The pICH41264 extracts are stocked at -20°C
Aurélie
Ursula
Dounia
-𝝱-10 bacteria : 30µl
Ligation product (paro): 4µl (insert or negative control)
30min_ 4°C
55 sec_ 42°C
2 min_ 4°C
+700µL of LB medium
1h_ 37°C
Staggering of 350 on LB+spec+Xgal dish
37°C O/N
Resuspension of IDT1 3’LTR in 31,03µL
Resuspension of IDT2 5’LTR in 27,56µL
[C]= 100 fmol/µL
Aurélie
Dounia
𝝱-10 Bacteria _ 30µL
Ligation product_ 4µL (insert or control)
- 30 min_4°c
- 55 sec_ 42°C
- 2 min _ 4°C
+ 700µl of LB
Spreading of 250µl on a dish containing LB + SPEC+ Xgal at 37°C
Aurélie
Ursula
| LTR3’1 | LTR5’1 |
| LTR3’2 | 2LTR5’2 |
| LTR3’3 | LTR5’3 |
| LTR3’4 | LTR5’4 |
Purification of plasmids (methode for digestion of verification)*? , using Nucleospin Plasmid Kit
1) Dilution at 10fmol/µl of each fragment received
| Fragment received | volume of H2O mQ to add (µl) |
|---|---|
| IDT3_L0_B1 | 1578/10 ⇒ 157,8 |
| IDT4_L0_B2 | 3216/10⇒321,6 |
| IDT6_L0_B3 | 900/10 ⇒ 90 |
| IDT4_L0_A3 | 1098/10⇒109,8 |
| IDT4_L0_A1 | 2078/10⇒207,8 |
| IDT4_L0_A2 | 1171/10⇒ 117,1 |
2) Reaction of digestion/ligation
Aurélie
Ursula
- 30µl of 𝝱+ 4µl of ligation product
- 30min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
- + 700µL of LB
Spreading of 250µL on 2 petri dishes Xgal+ Spec : control/ Gagpol
Incubation at 30°C over night
Ursula
Asmaa
Purification of PAGM9121 plasmide using Nucleopsin Plasmid Kit
| Fragment | Concentration (ng/µl) | ||
|---|---|---|---|
| LTR3’1 | 42,2 | 1.97 | 1.85 |
| LTR3’2 | 38,4 | 1.98 | 1.86 |
| LTR3’3 | 37,7 | 1.95 | 1.64 |
| LTR3’4 | 94,1 | 1.91 | 2.06 |
| LTR5’1 | 48,4 | 1.95 | 1.86 |
| LTR5’3 | 120 | 1.89 | 1.94 |
| Paromycin 1 | 55,8 | 1.91 | 1.89 |
| Paromycin 2 | 49,1 | 1.91 | 1.87 |
| GAG_Pol 1 | 78,7 | 1.89 | 1.92 |
| GAG_Pol 2 | 25,3 | 1.97 | 2.85 |
| GAG_Pol 3 | 49,0 | 1.94 | 2.62 |
| GAG_Pol 4 | 17,7 | 2.00 | 3.90 |
| GAG_Pol 5 | 44,0 | 1.97 | 2.68 |
| GAG_Pol 6 | 21,2 | 2.01 | 3.65 |
| Component | 10 µl reaction |
|---|---|
| DNA | 200 ng (except for GAG pol 4 & 6 : DNA=150ng) |
| 10X CutSmart Buffer | 1µl |
| BsaI | 0.2µ |
| Nuclease-free Water | to 50 µl |
Aurélie
Anaïs
Figure : Gel electrophoresis of digestion reaction products to check the inserts size
Agarose gel 1%
2µL loading buffer 6X per 10µl sample + BET
Migration 100V, 20 min
Aurélie
Anaïs
[C] Nanodrop
PSAD 2a: 107.9 ng/µL
1.97
3.81
PSAD 2d: 150.6 ng/µl
1.94
3.12
Intron Rb= 103 ng/µL
1.99
3.82
[AMG 9121]: 71.5 ng/µl
Samples sent to sequencing
Aurélie
Anaïs
| Fragment | Comments |
|---|---|
| Paromycine | ok |
| Gagpol (GG40 and GG41) Rubisco intron pSAD |
Absurd results Re-do the digestion reaction whith different BsaI enzymes |
Aurélie
Anaïs
- 10µL of 𝝱10 bacterias
- Gag Pol 5 : 1.5 µL
Gag Pol 3 : 0.5 µL
Igem 2, 4 and 6: 2µL
Igem 3, 2 : 2.5 µL
↳ plasmids to transfect
- 30 min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
+ 700 µl of LB
Spreading of 250µL on plates containing LB+ spec
Aurélie
Dounia
Kit used = Nucleopsin Plasmid
Elution volum= 40µL
| Fragment | Concentration(ng/µl) | ||
|---|---|---|---|
| LTR3’2 | 241.3 | 1.90 | 2.17 |
| ParoII | 196.0 | 1.90 | 2.09 |
| LTR5’ | 166.2 | 1.89 | 2.15 |
| ParoI | 160.9 | 1.91 | 2.11 |
| LTR3’4 | 232.8 | 1.89 | 2.21 |
| Gag5 | 393.6 | 1.91 | 2.16 |
| Rubisco | 31.3 | 2.04 | 2.09 |
| pSAD | 133.8 | 1.90 | 2.12 |
| Gag3 | 370.6 | 1.91 | 2.11 |
Aurélie
Anais
Put on liquid media the 24/07
kit used : Nucleopsin plasmid
| Fragment | Concentration(ng/µl) | ||
|---|---|---|---|
| Paro II | 110.4 | 1.89 | 2.21 |
| PSAD | 62.1 | 1.89 | 2.21 |
| GagPol 5 | 231.6 | 1.89 | 2.21 |
| ParoI | 96.4 | 1.89 | 2.18 |
| LTR5’ | 65.4 | 1.90 | 2.17 |
| LTR3’2 | 102.3 | 1.91 | 2.26 |
| LTR3’4 | 111.0 | 1.88 | 2.29 |
| Gag Pol3 | 189.8 | 1.90 | 2.21 |
| Intron Rubisco | 73.9 | 1.89 | 2.19 |
| Intron | PSAD | LTR5’1 | LTR3’4 | LTR3’2 | Uni | GagPol3 | GagPol5 | |
|---|---|---|---|---|---|---|---|---|
| Enzyme | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.2 | 0.2 |
| Buffer | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| ADN | 2 | 2 | 4 | 2 | 5 | 3 | 3 | 3 |
| H2O | 6.8 | 6.8 | 4.9 | 6.8 | 3.9 | 5.9 | 5.9 | 5.9 |
| BstEII SaeI |
BstEI StuI |
BstEII BamHI |
BstEII BamHI |
BstEII BamHI |
BstEII BamHI |
BstEII | BstEII |
Digestion at 37°C/ 1H , conservation at -20°C overnight
Aurélie
Anais
1% agarose gel, deposit of 10µ+2µL of loading buffer 6X, ladder “2log”
Gel plan
| Rb intron | pSAD | LTR3’2 | LTR3’4 | LTR5’4 | 9121 | Gag3 | Gag5 |
|---|---|---|---|---|---|---|---|
| 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 | 1 3 | 1 3 |
| 3 bands 1714 pb 620 pb 66 pb |
2 bands 1662 pb 1182 pb |
1 band 2000-3000pb (2270 pb) |
1 band 2270 pb |
2 band 11831pb 828 pb |
2 bands 4242 pb 2260 pb |
||
Figure : Gel electrophoresis of digestion reaction products to check the inserts size
There seems to be no inserts
- NDE I, 1h, 37°C
- Tampon 10X : 1µl
- ADN : 3µL
- Nde I : 0.2 µL
- H2O : 5.8 µ
| Rb Intron | pSAD | LTR3’2 | LTR3’4 | LTR5’1 | GagP3 | Gag P5 | Paro I | Paro II |
|---|---|---|---|---|---|---|---|---|
| 1 2 | 1 2 | 1 2 | 1 2 | 1 2 | 1 | 1 | 1 2 | 1 2 |
Non informative, forgotten time of incubation
Aurélie
| 1 reaction | mix *20 | |
|---|---|---|
| Bcl Enzyme | 0.2 µL | 4 µL |
| Buffer FD (10X) | 1 µL | 20µL |
| DNA | 3 µL | ↗ |
| H2O | 5.8 µL | 116 µL |
↪7µl mix/ per tube
+ 3µL DNA
+ 0.2 µl XmaII in pSAD
Incubation 2H at 37°C
Migration in 0.1 % agarose gel, ladder “2log”, deposit of 10µL of reaction sample + 2µl de loading buffer 6X
Aurélie
pSAD
AYL0014041 ← GG40
042 ← GG41
| 1 reaction | mix *12 | |
|---|---|---|
| Bcl Enzyme | 0.2 µL | 24 µL |
| Buffer Cutsmart (10X) | 1 µL | 12µL |
| DNA | 4 µL | ↗ |
| H2O mq | 4.8 µL | 57.6µL |
Incubation 3h 37°C
Migration on 1% agarose gel of 10µl sample + 2µL loading buffer
Ladder 2Log
90V, 20 minutes
OK for gag pol 3’ and 5’ LTR, Rb+ paro Sequencing
pSAD not ok → replanting of colonies
Aurélie
- GAG 3 (10µL DNA+ 10µL H2O)
- GAG 5 (10µL DNA+ 10µL H2O)
- 3’ LTR (3’4) (10µL DNA+ 5 µL H2O)
- 5’ LTR (10µL DNA+ 5 µL H2O)
- Rb (10µL DNA+ 5 µL H2O)
- GG40 (50µL) and GG41 (10µL)
-Ig18 (20µL), Ig19, Ig20 (10µL)
Stock from 07/25/18
Aurélie
Replanting of PSAD and Rb intron in liquid media+ spec .
Aurélie
Miniprep of PSAD and Rb (5 clones of each)
Kit used: Nucleopsin Plasmid
Aurélie
| 1 reaction | mix *12 | |
|---|---|---|
| BSAI Enzyme | 0.2 µL | 2.4 µL |
| Buffer Cut Smart (10X) | 1 µL | 12µL |
| DNA | 4 µL | ↗ |
| H2O mq | 4.8 µL | 57.6µL |
6µl of mix per tube + 4µl of DNA
Digestion 2H at 37°C+ 2µL of loading buffer 6X, 4µL of 2log ladder
Migration 90V, 40min
Figure : Gel electrophoresis of digestion reaction products to check the inserts size : 5 new clones of pSAD and Rb
Aurélie
-90µl of DH5α +Ligation product +10µl CMR
30 min 4°C
90 sec at 42°C
2 min on ice
700µL LB
1h at 37°C
spreading on 2 dishes
40mg ⇨ 2mL DMSO
Aurélie
Ursula
Reaction Mix
| Solutions | Volume for 1 tube (uL) | Volume for 7 tubes (uL) |
|---|---|---|
| Plasmid DNA | 1 | - |
| Phusion Enzyme | 0.25 | 1.75 |
| HF Buffer (5X) | 5 | 35 |
| dNTP | 0.5 | 3.5 |
| Forward Primer | 1.25 | - |
| Reverse Primer | 1.25 | - |
| H2O | 15.75 µL | 110.25 |
- 21.5 uL of Mix/Tube
- 1 uL of DNA
- 2 X 1.25 uL Primer
pSAD = Ig3/Ig4 primers + template p0.07 → 807 bp
Rb intron = Ig5/Ig6 primers + template 0.07 → 177 bp
1- p007 + Ig3/Ig4
2- pUC19 + Ig3/Ig4
3- H2O + Ig3/Ig4
4- p0-02 + Ig5/Ig6
5- pUC19 + Ig5/Ig6
6- H2O + Ig5/Ig6
Aurélie
Ursula
Products of the PCR à OK
IDT4 L0A1 + IDT4 L0A2 + IDT4 L0A3 + IDT3 L0B1 à OK
No bands for IDT4 L0B2 Were the samples mixed during the loading?
Plusieurs bands for IDT6 L0B3
Elution in 20 uL
Used kit: Nucleospin Gel and PCR clean up
Concentration measurements using the Nanodrop:
- PSAD= 39.8 ng/uL
- Rb= 55.1 ng/uL
Aurélie
Soukeyna
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
Rb 1 : 109.9 ng/µl
Rb 2 : 96.8 ng/µl
Rb 3 : 77.2 ng/µl
Rb 4 : 88.7 ng/µl
Digestion of the plasmid by BstEII and SalI
Goal : Check if the plasmid effectively contain the insert “Rubisco intron”
Expected result :
| Lader (2log) | Rb1+ | Rb2+ | Rb3+ | Rb4* |
|---|
Figure : Gel electrophoresis of digestion reaction products to check the inserts size
add 2 µl of primers at 10 µL for each tube
Provider : DC biosciences
| Part | DNA concentration (ng/µl) |
|---|---|
| pUC-SP VS7 otsA_B21 | 165.3 |
| pUC-SP VS8 otsA_B22 | 103.9 |
| pUC-SP VS9 otsA_B23 | 107.6 |
| pUC-SP VS10 otsB_B41 | 242.7 |
| pUC-SP VS11 otsB_B42 | 81.2 |
Aurélie
Ursula
Add 2µl of primers at 10µM
Aurélie
Soukeyna
- 25µl of B10 bacteria + 4µl of ligation product (4 different transformations)
- 30 min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
+700µl of LB medium
1 h at 37°C
Spreading of 250 µl in plates containing LB+spectinomomycin+X-gal
kit used: Machinery Nagel easypure kit plasmid DNA extraction
a : 52.8 ng/µl 1.91 1.98
b : 39.8 ng/µl 1.90 1.84
2 clones of OTSA : OTSA1 OTSA2 (one culture per clone)
2 clones of OTSB : OTSB1 OTSB2 (one culture per clone)
1 clone of pGAM 1276
1 clone of pAGM1299
the 6 cultures previously described are placed on a will for over week/end culture
Aurélie
Then, hybridation during 15 minutes (there are in fact, the missing DNA fragment of GAGPOL) .
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
- pAGM1276 : 56ng/µl
- pAGM1299 : 76.6ng/µl
- otsA 1 : 78.4ng/µl
- otsA 2 : 75.6ng/µl
- otsB 1 : 73.1ng/µL
- otsB2 : 78.2ng/µl
Aurélie
Soukeyna
Digestion reaction of otsA :
- bstEII-HF : 0.2µl
- CutSmart buffer (10X) : 1 µl
- DNA plasmid : 3µl
- H20: 5.8µl
Digestion reaction of otsB :
- bstEII-HF : 0.1µl
- NotI-HF : 0.1µl
- CutSmart buffer (10X) : 1µl
- DNA plasmid : 3µl
- H20 : 5.8µl
Electrophoresis on 0.1% agarose, with 2 log ladder and 12µl of reaction
Figure : Gel electrophoresis of digestion reaction products to check the inserts size
The band on the gel are correct but there is a lot od DNA on the top of the gel and the secnd band is very weak. Morover, the sequence we ordered for otsB is wrong, so we need to correct it by PCR. So we will ordered primers and we’ll send to sequence after the PCR. For otsA, we’ll make an other transformation to select only one pure clone
25µl of B10 bacteria + 4µl of ligation product (4 different transformations)
30 min at 4°C
55 sec at 42°C
2 min at 4°C
+700µl of LB medium
1 h at 37°C
Spreading of 250 µl in plates containing LB+spectinomomycin+X-gal for p0-iGEM5
Spreading of 250 µl in plates containing LB+ampicilin+X-gal for p1-iGEM2
Soukeyna
Observation of plates from the day before
- p1-iGEM2 (Cargo) : only blue colonies for + and - controle
- p0-iGEM5- GagPol: only blue colonies for the positive (+) experiment , and both white and blue colonies for the negative (-) control
Composition of the media :Amp+ Spec+ Xgal
4 colonies were replanted
Aurélie
Soukeyna
Kit utilisé : NucleoSpin Plasmid (Macherey-Nagel)
p0-iGEM5-1 : 97.8ng/µl
p0-iGEM5-2 : 89.5ng/µl
p0-iGEM5-3 : 123.4ng/µl
p0-iGEM5-4 : 67.6ng/µL
| Clone 1 and 3 | Clone 2 and 4 | |
|---|---|---|
| bstEII enzyme | 0.2µl | 0.2µl |
| Plasmid DNA | 2µl | 3µ |
| NEB cutsmart buffer (10X) | 1µl | 1µ |
| H20 | 6.8µl | 5.8µl |
Figure : Gel electrophoresis of digestion reaction products to check the inserts size
25µl of bacteria + 3µl of ligation product
30min at 4°C
55sec at 42°C
2min at 4°C
+700µl of LB medium on each tube
1h at 37°C
Spreading of 200µl of each transformation on LB + Xgal + amp + IPTG plate
Oovernight at 37°C
Aurélie
- p0-iGEM8 (otsB) : 1µl at 2ng/µl
- Enzyme Phusion : 0.25µl
- HF Buffer (5X) : 5 µl
- dNTP : 0.5µl
- Primer OTSB-v2-F : 1.25 µl
- Primer OTSB-v2-R : 1.25µl
-H20 : 15.75 µl
| *30 | |||||
|---|---|---|---|---|---|
| 98°C | 98°C | 50°C | 72°C | 72°C | 12°C |
| 1min | 1 min | 2 min | 3 min | 3 min | Hold |
Aurélie
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
#1 : 101.5ng/µl
#2 : 90.0ng/µl
#3 : 97.7ng/µL
#4 : 75.1ng/µl
#5 : 61.5ng/µl
#6 : 115.6ng/µl
| NcoI | 0.1µl | 6.5µl |
|---|---|---|
| NdeI | 0.1µl | 6.5µL |
| NotI | 0.1µl | 6.5µl |
| Cutsmart buffer 10X | 1µl | 6.5µl |
| ADN | 2.5µl | |
| H2O | 6.2µl | 40.3µl |
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
7.5µl of mix by tube + 2.5µl of DNA
On 1% agarose gel, with otsB PCR from 7/09 : expected result : >800bp
B10 bacteria + 3.5µl of ligation product
- 30 min at 4°C
- 55sec at 42°C
- 2min at 4°C
- +700µl of LB medium
- 1h at 37°C
- Spreading of 200µl on plate with LB+spec+Xgal
Sara and Parisa
| Components | 1 reaction | Mix (*6) |
|---|---|---|
| bbsI | 0.5µL | 3 µL |
| Cutsmart buffer (10X) | 1 µL | 6 µL |
| DNA | 2.5 µL | 6.5µl |
| H2O | 6 µL | 36µL |
2 white colonies were observed on the plate containing bacterias transformed with GAGPOL
- Replanting on liquid media
- Replanting on plate (not isolable)
Sara and Parisa
Victor
(0.1% agarose gel, 2-log ladder, 100V)
We were expecting a 4.5kb band for the plasmid and a 2.6kb band for the insert.
We do have the expected band for the plasmid but, we observe a band at 0.6kb for the insert (Lac Z maybe?)
Kit used Nucleospin Plasmid
Kit used : Nucleospin gel to PCR clean up
Insert OtsB= 4ng/µL
GAG palamsid = 48.1 ng/µl
Insert OtsB= 4ng/µL
GAG palamsid = 48.1 ng/µl
→ purification of PCR band and Launch of miniprep
(To do next level 1 pI-Igem2)
Sara
Victor
𝛃10+ 4µL of OtsB ligation reaction products
- 30min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
- Addition of 70µL of LB
1 Hour at 37°C
Spreading of 200µL on LB+Xgal + Spec plate
37°C O/N
Kit used Nucleospin Plasmid
26.4 ng/µL
bbsI =0.5µL
buffer= 2µL
DNA =10µL
H2O= 6.5µL
Expected result: 4,4kb + (?)
ast well = digestion reaction product of GagPOL from the day before → Does not correspond to the expected lenght
Sara
Victor
3 ligation tubes are prepared , T-, G, pre-mix of E, T-
- Tube 1 : T- 1.2µl of blackbone +12.8 µL of H2O (14µL in total)
- Tube 2 : G 2 µL of the 6 sub-parts for GagPol)+.0.8µl of H2O +1.2µL of backbone ( 14µl in total)
Tube 3: Master mix of enzymes
| Components | 1 reaction |
|---|---|
| ATP | 4 µL |
| Cutsmart buffer (10X) | 4 µL |
| T4 ligase | 2 µL |
| bbstI | 2 µL |
12µL of mix in total dispatched in tubes 1 and 2 (6µL each)
- 0.5g of agarose
- 50ml of TEA 1X
- Cyber green
Gel plan: well 1 : 3µl of DNA ladder, well 2 and 3 10µl of Gagpol
For each :
- 4ml of LB media
- 2µl of spectinomycin (100mg/ml) (in order to obtain 10µg/ml)
- 1 white clone
Incubation at 37°C overnight
- Otsb miniprep + digestion
- Gag digestion
- Gag transfo)
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