Difference between revisions of "Team:Sorbonne U Paris/Notebook"
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<td class="td-notebook">HF buffer (5X)</td> | <td class="td-notebook">HF buffer (5X)</td> | ||
<td class="td-notebook">5</td> | <td class="td-notebook">5</td> | ||
| − | <td class="td-notebook">40</td> | + | <td class="td-notebook"">40</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 437: | Line 437: | ||
<tr> | <tr> | ||
<td class="td-notebook"> Rv Primer (10µM) </td> | <td class="td-notebook"> Rv Primer (10µM) </td> | ||
| − | <td class="td-notebook>1.25</td> | + | <td class="td-notebook">1.25</td> |
<td class="td-notebook"></td> | <td class="td-notebook"></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td class="td-notebook>H20</td> | + | <td class="td-notebook">H20</td> |
<td class="td-notebook">15.75</td> | <td class="td-notebook">15.75</td> | ||
<td class="td-notebook">126</td> | <td class="td-notebook">126</td> | ||
Revision as of 11:25, 29 September 2018
Notebook
Calendars 2018
| 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 | |||||
June
Tuesday 12th June 2018
PCR of rubisco intron, paromomycin CDS and pSAD promoter
Institut de biologie physico-chimique (IBPC)
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
- negative control : puc 19 + Ig5/Ig6
- Rubisco intron : p0-02 + Ig5/Ig6
- negative control : puc 19 + Ig3/Ig4
- pSAD promoter : p0-07 + Ig3/Ig4
- negative control : puc 19 + Ig11/Ig12
- paromomycin CDS : p0-12 + Ig11/Ig12
- negative control : H20 + Ig5/Ig6
- negative control : H20 + Ig11/Ig12
Cycle : 98°C, 35*(98°C - 1min, 50°C - 2min, 72°C - 3min), 72°C - 3min, 12°C - hold
Wednesday 13th June
Migration and purification of PCR product
“paromomycin”, “pSAD”, “rubisco intron”
Institut de biologie physico-chimique (IBPC)
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 |
Gel analysis
Figure 1 : Gel electrophoresis of PCR products rubisco (2&3) intron,pSAD (4&5), paromomycin(6&7)
Rubisco and pSAD : Ok for rubisco intron and pSAD, visible bands
Paromycin: the main band does not have the expected length, the band at 1.6 kb is still purifed
Cutting of the bands corresponding to the expected bands (photobenchling)
DNA purification
Kit used: Nucleospin Gel 2 PCR Clean up- gel extraction (NT1= 800µl)Elution in 15µl , mesure of the DNA extracts concentration using a nanodrop
| 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”
Launch of 2 bacterial cultures overnight (4ml with spectinomycine)
- p0universal = pAGM9121
- p0B3 pICH41258
Wednesday 14th June
Plasmid purification & Reaction of ligation/digestion
Institut de biologie physico-chimique (IBPC)
Victor
Aurélie
Soukeyna
Aim : Extraction of plasmides from yesterday’s cultures and reactions of digestion/ligation p0universal/ pSAD and p0-B3/intron rubisco.
Plasmid purification
(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
Reaction of ligation/digestion
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- ∞
Wednesday 20th June
PCR of the “ paromycine” fragment
Purification of “p0- IGEM1” and “p0- IGEM3” plasmides
Muséum national d'histoire naturelle (MNHN)
Aurélie
Ursula
PCR reactions
| 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
Purification of “p⦽-IGEM1” and “p⦽-IGEM3” plasmids
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
Wednesday 27th June
Bacterial culture
Muséum national d'histoire naturelle (MNHN)
Aurélie
Ursula
Launch of 2* 3.5 mL of bacterial culture → +spectromycine of pICH4 1264 which contains the destination plasmid of the “paromicycn fragment)
Wednesday 29th June
Purification of pICH41264 plasmid using the Nucleospin Plasmid K
Plasmid concentration measures using nanodrop
Muséum national d'histoire naturelle (MNHN)
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
july
Monday 2 July
Transformation of 𝝱-10 bacterias with p⦽ pICH4 1264 + insert paroR
Production of p⦽ plasmids for 3’LTR and 5’LTR parts.
Institut de biologie physico-chimique (IBPC)
Aurélie
Ursula
Dounia
Transformation of 𝝱-10 bacterias with p⦽ pICH4 1264 + insert paroR
-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
Production of p⦽ plasmids for 3’LTR and 5’LTR parts.
Resuspension of IDT1 3’LTR in 31,03µL
Resuspension of IDT2 5’LTR in 27,56µL
[C]= 100 fmol/µL
Thurday 5 July
Transplanting of single clones from each subcloning of the 3rd of july
Transformation of 𝝱-10 bacterias with p⦽ pUniversal, containing either LTR5’ or LTR3’ as insert
Institut de biologie physico-chimique (IBPC)
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
Thurday 9 July
Lauch of miniprep
Verifcation of Paro1 and Paro 2 clones
Assembly of GAG-Pol
Institut de biologie physico-chimique (IBPC)
Aurélie
Ursula
Lauch of miniprep of 4 clones of each LTD in media spec+ Xgal
| LTR3’1 | LTR5’1 |
| LTR3’2 | 2LTR5’2 |
| LTR3’3 | LTR5’3 |
| LTR3’4 | LTR5’4 |
Verifcation of Paro1 and Paro 2 clones
Purification of plasmids (methode for digestion of verification)*? , using Nucleospin Plasmid Kit
Assembly of GAG-Pol ⇨ 6 parts
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
Thurday 10th July
Transformation of 𝝱10 with digestion/ligation products of GAG POL
(experiences of 07/09/18)
Institut de biologie physico-chimique (IBPC)
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
Friday 13th July
Launch of minipreps for 6 clones of GAG-Pol
Institut de biologie physico-chimique (IBPC)
Ursula
Asmaa
Launch of minipreps for 6 clones of GAG-Pol in Xgal+spec media GAG_Pol 1, GAG_Pol 2, GAG_Pol 3, GAG_Pol 4 , GAG_Pol 5, GAG_Pol 6
Purification of PAGM9121 plasmide using Nucleopsin Plasmid Kit
Measure of DNA concentration at Nanodrop
| 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 |
Digestion to check the inserts size ( using BsaI)
| 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 |
Monday 16th July
Migration of digestion reaction products
Institut de biologie physico-chimique (IBPC)
Aurélie
Anaïs
Migration of digestion reaction products from the day before
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
Tuesday 17th July
Arrangement in the box iGEM 2018
Preparation of all the inserts in order to send them to sequencing
Institut de biologie physico-chimique (IBPC)
Aurélie
Anaïs
Arrangement in the box iGEM 2018
Preparation of all the inserts in order to send them to sequecing
[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
Thurday 19th July
Analysis of sequencing results
Institut de biologie physico-chimique (IBPC)
Aurélie
Anaïs
| Fragment | Comments |
|---|---|
| Paromycine | ok |
| Gagpol (GG40 and GG41) Rubisco intron pSAD |
Absurd results Re-do the digestion reaction whith different BsaI enzymes |
Monday 23th July
Transformation to redo the stock of each plasmid
Institut de biologie physico-chimique (IBPC)
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
Thurday 24th July
Transplanting of a clamydomonas clone
Plasmid extraction from 𝝱 bacterias transformed
Concentration measurements
Institut de biologie physico-chimique (IBPC)
Aurélie
Dounia
1. Transplanting of a clamydomonas clone in 3ml of LB+ Spec
2. Plasmid extraction from 𝝱 bacterias transformed the day before ( directly from dish).
Kit used = Nucleopsin Plasmid
Elution volum= 40µL
3. Concentration measurements using Nanodrop
| 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 |
4. Replanting of Chlamydomonas
Wednesday 25th July
Plasmid extraction from bacteria transformed
Concentration measurement
Digestion
Institut de biologie physico-chimique (IBPC)
Aurélie
Anais
Plasmid extraction from bacteria transformed
Put on liquid media the 24/07
kit used : Nucleopsin plasmid
Concentration measurement using Nanodrop
| 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 |
Digestion
| 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
Thurday 26th July
Migration of digestion samples
Digestion
Migration on gel electrophoresis
Muséum national d'histoire naturelle (MNHN)
Aurélie
Anais
Migration of digestion samples from the day before
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
Digestion
- NDE I, 1h, 37°C
- Tampon 10X : 1µl
- ADN : 3µL
- Nde I : 0.2 µL
- H2O : 5.8 µ
Migration on gel
| 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
Friday 27th July
Enzymatic digestion reaction
Muséum national d'histoire naturelle (MNHN)
Aurélie
Enzymatic digestion reaction ( fast digest enzyme)
| 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
Tuesday 31th July
DNA sent to sequencage
Digestion reaction using BSaI
Muséum national d'histoire naturelle (MNHN)
Aurélie
1. DNA sent to sequencage
pSAD
AYL0014041 ← GG40
042 ← GG41
Digestion reaction using BSaI
| 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
August
Wednesday 1st August
Sending samples to sequencing
Cloning of pSAD
Muséum national d'histoire naturelle (MNHN)
Aurélie
1. Sending samples to sequencing
- 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
Cloning of pSAD from 07/23/2018 experiment, in solid LB+ spec
Thurday 2nd August
Replanting of PSAD and Rb intron
Muséum national d'histoire naturelle (MNHN)
Aurélie
Replanting of PSAD and Rb intron in liquid media+ spec .
Friday 3th August
Miniprep of PSAD and Rb
Muséum national d'histoire naturelle (MNHN)
Aurélie
Miniprep of PSAD and Rb (5 clones of each)
Kit used: Nucleopsin Plasmid
Monday 6th August
Digestion of the 5 new clones of pSAD and Rb
Muséum national d'histoire naturelle (MNHN)
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
Tuesday 7th August
Digestion/ ligation reaction of pSAD 2 Rb intron
Transformation
Preparation X-GAL
Aurélie
1. Digestion/ ligation reaction of pSAD 2 Rb intron
see 06/14/2018 protocol
2. Transformation
-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
3. Preparation X-GAL
40mg ⇨ 2mL DMSO
Tuesday 21th August
New PCR of “pSAD” and “rubisco” intron
Institut de biologie physico-chimique (IBPC)
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
Tuesday 22th August
Migration of the PCR products and the fragments of GAG Pol
Purification of the product the gel of the 2 PCRs:
Cloning of pSAD
Institut de biologie physico-chimique (IBPC)
Aurélie
Ursula
1. Migration of the PCR products and the fragments of GAG Pol sent by IDT
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
2.Purification of the product the gel of the 2 PCRs
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
3. Cloning of pSAD
Intron Rubisco
Ligation
Cycle
Tuesday 28th August
Plasmidic extraction of bacteria transformed
Measure of the concentration
Digestion of the plasmid by BstEII and SalI
Preparation of DNA samples for sequencing
Institut de biologie physico-chimique (IBPC)
Aurélie
Soukeyna
Plasmidic extraction of bacteria transformed by plasmid containing “Rubisco intron”
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
Measure of the concentration at Nanodrop
Rb 1 : 109.9 ng/µl
Rb 2 : 96.8 ng/µl
Rb 3 : 77.2 ng/µl
Rb 4 : 88.7 ng/µl
Measure of the concentration at Nanodrop
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
Preparation of DNA samples for sequencing
add 2 µl of primers at 10 µL for each tube
Concentration measurement on Nanodrop of recieved parts for trehalose
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 |
Tuesday 29th August
Sample preparation to send “rubisco intro” at sequencing
Institut de biologie physico-chimique (IBPC)
Aurélie
Ursula
Add 2µl of primers at 10µM
Tuesday 30th August
Digestion/ligation reaction of otsA and otsB
Transformation of B10 bacteria with ligation product
PlCH47811 plasmid extraction
Replanting of 𝝱10 clones transformed
Checking of DNA content of IDT4 sample for Gag Po
Institut de biologie physico-chimique (IBPC)
Aurélie
Soukeyna
1. Digestion/ligation reaction of otsA and otsB
2. Transformation of B10 bacteria with ligation product
- 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
3. PlCH47811 plasmid extraction from overnight culture
kit used: Machinery Nagel easypure kit plasmid DNA extraction
4. PlCH47811 plasmid DNA concentration measurement on Nanodrop
a : 52.8 ng/µl 1.91 1.98
b : 39.8 ng/µl 1.90 1.84
5. Replanting of 𝝱10 clones transformed the day before on LB media + spectinomycine
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
6. Checking of DNA content of IDT4 sample for Gag Pol
The tube is vortexed upside down then centrifuged (to check if the DNA is not stuck on the tube’s lid)DNA concentration measurement on nanodrop:
(blank : H2O)
1.5ng/µL
1.6ng/µL
1.5ng/µl
September
Monday 3th September
Suspension of primers IDT-4L0-B2-F and IDT-4L0-B2-R
Miniprep of otsA, otsB and pAGM1276 and pAGM1299
Concentration measurement
Level 0 ligation protocol for GAGPOL parts and Level 1 ligation protocol for the CARGO retrotransposon
Institut de biologie physico-chimique (IBPC)
Aurélie
1. Suspension of primers IDT-4L0-B2-F and IDT-4L0-B2-R received today according to Eurofins instructions
Then, hybridation during 15 minutes (there are in fact, the missing DNA fragment of GAGPOL) .
2. Miniprep on the over week-end culture of otsA, otsB and pAGM1276 and pAGM1299
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
3. Concentration measurement at Nanodrop
- 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
4. Level 0 ligation protocol for GAGPOL parts : (see 09/07/2018)
5. Level 1 ligation protocol for the CARGO retrotransposon
Tuesday 4th September
Digestion of otsA and otsB ligation reaction
Transformation of B10 bacteria with cloning reaction
Institut de biologie physico-chimique (IBPC)
Aurélie
Soukeyna
1. Digestion of otsA and otsB ligation reaction of 30/08
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
2. Transformation of B10 bacteria with cloning reaction of 03/09 : p1-iGEM1 and p0-iGEM5
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
Wednesday 5th September
PCR amplificication
Institut de biologie physico-chimique (IBPC)
Soukeyna
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Tuesday 6th September
PDNA extraction of 4 differents clones p0-iGEM5
Concentration measurement
Verification digestion of p0-iGEM5 plasmid
Transformation of bacteria with the ligation produc
Institut de biologie physico-chimique (IBPC)
Aurélie
Soukeyna
1. DNA extraction of 4 differents clones p0-iGEM5
Kit utilisé : NucleoSpin Plasmid (Macherey-Nagel)
2. Concentration measurement at Nanodrop
p0-iGEM5-1 : 97.8ng/µl
p0-iGEM5-2 : 89.5ng/µl
p0-iGEM5-3 : 123.4ng/µl
p0-iGEM5-4 : 67.6ng/µL
3.Verification digestion of p0-iGEM5 plasmid
| 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
4. Transformation of bacteria with the ligation product of the 05/09
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
Friday 7th September
Ligation reaction of p0-iGEM5 (GAGPOL)
otsB PCR
Institut de biologie physico-chimique (IBPC)
Aurélie
1. Ligation reaction of p0-iGEM5 (GAGPOL)
2. otsB PCR
- 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 |
3. 6 colonies from yesterday transformation p1igem2 in LB medium + amp
Monday 10th September
DNA extraction of the 6 bacteria culture
Concentrations measurement
Digestion reaction to check p1-iGEM2 cloning reaction
Transformation of B10 bacteria
Institut de biologie physico-chimique (IBPC)
Aurélie
1. DNA extraction of the 6 bacteria culture prepare on 7/09
Used kit : NucleoSpin Plasmid (Macherey-Nagel)
2. Concentrations measurement at Nanodrop
#1 : 101.5ng/µl
#2 : 90.0ng/µl
#3 : 97.7ng/µL
#4 : 75.1ng/µl
#5 : 61.5ng/µl
#6 : 115.6ng/µl
3. Digestion reaction to check p1-iGEM2 cloning reaction
| 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)
Concentrations measurement at Nanodrop
7.5µl of mix by tube + 2.5µl of DNA
On 1% agarose gel, with otsB PCR from 7/09 : expected result : >800bp
4. Transformation of B10 bacteria with ligation product from 07/09
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
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