Team:Lethbridge/Notebook/Lab Work/August
From 2010.igem.org
(→August 3/2010 Evening) |
(→August 4/2010) |
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15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)<br> | 15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)<br> | ||
- | ==August 4 | + | ==August 4, 2010== |
- | (in | + | (in lab: JV)<br> |
- | <b> Objective:</B> PCR analysis of ligation product of | + | <b> Objective:</B> PCR analysis of ligation product of August 3, 2010<br> |
- | + | * <u>Ligations</u> | |
- | + | ||
- | * < | + | |
** pBAD-mRBS | ** pBAD-mRBS | ||
** pBAD-SRBS | ** pBAD-SRBS | ||
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** dt-pSBIC3 | ** dt-pSBIC3 | ||
** pLacI-SRBS | ** pLacI-SRBS | ||
- | + | * <u>Controls</u> | |
- | * < | + | |
** pBAD | ** pBAD | ||
** TetR | ** TetR | ||
** TetR | ** TetR | ||
** pLacI | ** pLacI | ||
- | ** | + | ** mms6 |
+ | <b>Method:</b><br> | ||
+ | <u>PCR:</u> Thermocycler set to iGEM program 7<br> | ||
<table border ="3"> | <table border ="3"> | ||
- | <tr><td> | + | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x16)(µL)</b> |
<tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>668.6 | <tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>668.6 | ||
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80 | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80 | ||
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<tr><td>Reverse Primers<td>0.5<td>8 | <tr><td>Reverse Primers<td>0.5<td>8 | ||
<tr><td>Template DNA<td>1<td>16 | <tr><td>Template DNA<td>1<td>16 | ||
- | <tr><td> | + | <tr><td>Pfu polymerase<td>0.2<td>3.2 |
</table><br> | </table><br> | ||
- | < | + | <u>2.5% agarose gel(1x TAE)</u><br> |
- | + | ||
<table border ="3"> | <table border ="3"> | ||
<tr><td>lane<td><b>contents</b><td><b>Successful Ligation ?</b> | <tr><td>lane<td><b>contents</b><td><b>Successful Ligation ?</b> | ||
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<tr><td>14<td>pBad-mRBS<td>x | <tr><td>14<td>pBad-mRBS<td>x | ||
<tr><td>15<td>pBad control<td>--- | <tr><td>15<td>pBad control<td>--- | ||
- | </table> | + | </table> |
+ | *Ran at 100V for 70 minutes. | ||
- | + | <b>Results:</b> <font color ="red">AGAROSE GEL PICTURE</font><br><br><br> | |
- | + | <b>Objective:</b> Transform the successful ligations<br> | |
- | <b>Objective:</b> Transform<br> | + | |
<b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol | <b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol | ||
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<font color ="red">"AUG 4" is not yet done</font> | <font color ="red">"AUG 4" is not yet done</font> | ||
- | |||
==August 4/2010== | ==August 4/2010== |
Revision as of 23:00, 30 August 2010
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Back to Notebook
Back to Lab Work
Contents |
August
August 3, 2010
(in Lab: HB, AV, JV)
Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used Restriction of Plasmid DNA protocol.
- A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
- pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
- A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
- pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
Construct | pDNA | buffer | Enzymes |
pBAD-sRBS/mRBS | pBAD | Red | EcoRI and SpeI |
pBAD-sRBS/mRBS | sRBS | Orange | XbaI and EcoRI |
pBAD-sRBS/mRBS | mRBS | Orange | XbaI and EcoRII |
sRBS/mRBS-TetR | sRBS | Red | PstI and SpeI |
sRBS/mRBS-TetR | mRBS | Red | PstI and SpeI |
sRBS/mRBS-TetR | TetR | Tango | XbaI and PstI |
TetR-dT | TetR | Red | EcoRI and SpeI |
TetR-dT | dT | Orange | XbaI and EcoRI |
dT-pTet | dT | Red | EcoRI and SpeI |
dT-pTet | pTet | Orange | XbaI and EcoRI |
pLAcI-sRBS/mRBS | pLacI | Red | EcoRI and SpeI |
pLAcI-sRBS/mRBS | sRBS | Orange | XbaI and EcoRI |
pLAcI-sRBS/mRBS | mRBS | Orange | XbaI and EcoRI |
Mms6-PET28(a) | PET28(a) | Orange | NotI |
- For all reactions
- 158 (µL) Milli-Q H2O
- 10 (µL) Buffer
- 0.5(µL) of each enzyme
- 10 (µL) pDNA
Restriction was incubated for 1 hour at 37oC
Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7
Component | Volume per tube (µL) | Master Mix (x6) |
MilliQ H2O | 41.8 | 250.8 |
10x Pfu Buffer with MgSO4 | 5 | 30 |
dNTPs | 1 | 6 |
Forward Primer | 0.5 | 3 |
Reverse Primer | 0.5 | 3 |
Template DNA | 1 | - |
Pfu DNA polymerase | 0.2 | - |
Total | 50 | 292.8 |
- Added 48.8 µL of Master Mix to each PCR reaction
Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:
Lane | Sample | Components (µL) |
1 | 1 kb ladder | 2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O |
2 | ECFP | 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O |
3 | EYFP | 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O |
4 | Lumazine | 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O |
- Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.
Results: AGAROSE GEL PICTURE
Objective: Ligate dT into pSB1C3.
Method:
- PCR amplify and purify both pSB1C3 and dT
- Restrict both with EcoRI and PstI
- Restrict pSB1C3 with DpnI
- Ligate pSB1C3 and dT
Restriction:
Restriction | MilliQ H2O (µL) | Buffer Orange (µL) | pDNA (µL) | Enzyme (µL) |
pSB1C3 | 79.25 | 10 | 10 | 0.25 EcoRI + 0.25 PstI + 0.25 DpnI |
pSB1C3 control | 80 | 10 | 10 | - |
dT | 79.50 | 10 | 10 | 0.25 EcoRI + 0.25 PstI |
dT control | 80 | 10 | 10 | - |
- Restriction were incubated at 37oC for 90 minutes.
- Enzymes were heat killed for 20 minutes at 80oC.
August 3, 2010 Evening
(in lab: KG, JS)
Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.
Lane | Contents | Result |
1 | 1kb ladder | |
2 | pTet (XbaI, EcoRI) | good |
3 | pTet (orange buffer) | --- |
4 | dT (XbaI, EcoR) | no good |
5 | dT (orange buffer) | --- |
6 | mRBS (SpeI, PstI) | good |
7 | mRBS (red buffer) | --- |
8 | sRBS (SpeI, PstI) | good |
9 | sRBS (red buffer) | --- |
10 | mRBS (XbaI, EcoRI) | good |
11 | mRBS (orange buffer) | --- |
12 | sRBS (XbaI, EcoRl) | good |
13 | sRBS (orange buffer) | --- |
14 | pet28(a) | good |
15 | 100 bp ladder | |
16 | PSB1C3 | not good |
17 | PSB1C3 restriction digest | --- |
Lane | Contents | Result |
1 | TetR (EcorI, SpeI) | good |
2 | TetR (red buffer) | --- |
3 | pLacI (EcoRI, SpeI) | good |
4 | pLacI (red buffer) | --- |
5 | Mms6 | can not tell |
6 | Mms6 control | --- |
7 | TetR (Xbal, PstI) | good |
8 | TetR (tango buffer) | --- |
9 | pBAD (EcoRI, SpeI) | good |
10 | pBAD (red buffer) | --- |
11 | dT (EcoRI, SpeI) | good |
12 | dT (red buffer) | --- |
13 | pLacI (2) | ? |
14 | dT control | not good |
15 | dT restriction | |
16 | 100 bp ladder | |
17 | dT PCR product | good |
18 | Mms6 PCR product | good |
19 | pBAD PCR product | good |
20 | pLacI PCR product | good |
Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3
Method: Ligation of Plasmid DNA
15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)
August 4, 2010
(in lab: JV)
Objective: PCR analysis of ligation product of August 3, 2010
- Ligations
- pBAD-mRBS
- pBAD-SRBS
- SRBS-tetR
- mRBS-TetR
- dt-pTet
- mms6-pET-28a
- dt-pSBIC3
- pLacI-SRBS
- Controls
- pBAD
- TetR
- TetR
- pLacI
- mms6
Method:
PCR: Thermocycler set to iGEM program 7
Component | 1X(µL) | Master Mix(x16)(µL) |
Milli-Q H2O | 41.8 | 668.6 |
10x Pfu Buffer with MgSO4 | 5 | 80 |
dNTPs | 1 | 16 |
Forward Primer | 0.5 | 8 |
Reverse Primers | 0.5 | 8 |
Template DNA | 1 | 16 |
Pfu polymerase | 0.2 | 3.2 |
2.5% agarose gel(1x TAE)
lane | contents | Successful Ligation ? |
1 | 50bp ladder | --- |
2 | dt pSBIC3 | --- |
3 | dt pTet | x |
4 | dt control | --- |
5 | sRBS-TetR | x |
6 | mRBS-TetR | ? |
7 | TetR control | --- |
8 | pLacI-mRBS | x |
9 | pLacI-sRBS | ? |
10 | pLacI control | --- |
11 | Mms6 pET-28a | no band |
12 | Mms6 control | --- |
13 | pBad-SRBS | x |
14 | pBad-mRBS | x |
15 | pBad control | --- |
- Ran at 100V for 70 minutes.
Results: AGAROSE GEL PICTURE
Objective: Transform the successful ligations
Method: used Competent Cell Transformation protocol
- changes:
- used 50µL aliquottes of DH5&alpha
- did not pipette up and down once, the cells were just swirled 3 times
- added 400µL SOC media, shoock at 370C for 90 min
- platted 250µL and 150µL
Incubated from 12:00AM to 4;00 PM
results | ||
contents | &250µL | 150µL |
dt-pTet | :) | x |
- control | x | x |
mms6-pET-28a | :) | :) |
dt-pSBIC3 | x | x |
mRBS-TetR | :) | :) |
pLacI-mRBS | :) | :) |
SRBS-TetR | x | x |
pBAD-SRBS | :) | :) |
+ contol | :) | :) |
"AUG 4" is not yet done
August 4/2010
Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.