Team:Lethbridge/Notebook/Lab Work/August
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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 | good | x |
- control | x | x |
mms6-pET-28a | good | good |
dt-pSBIC3 | x | x |
mRBS-TetR | good | good |
pLacI-mRBS | good | good |
SRBS-TetR | x | x |
pBAD-SRBS | good | good |
+ contol | good | good |
August 6/2010 Evening
Objective: Attempt colony pcr for rapid screening
Method: Followed two protocols from openwet
- Knight Protocol
- place 20(µL) sterile H2O in 0.6mL sterile tube
- with P10 pipette set to 3(µL) dip tip into colony
- place pipette tip into water and pipette up and down 20 times(this can be stored at 40C for inoculation of overnight 5mL cultures)
- Endy Protocol
- place 50(µL) sterile H2O in 0.6mL sterile tube
- with PLO pipette (set 3(µL)) dip sterile tip into colony
- place pipette tip into water and pipette up and down 20 times
Knight cont'd | Endy cont'd |
setup 20(µL) reaction | setup 20(µL) reaction |
1(µL) colony suspension | 2(µL) colony suspension |
2(µL) 10x p.fu (+Mg SO4 | 2(µL) 10x p.fu (+Mg SO4 |
2(µL) dNTP | 2(µL) dNTP |
1.25(µL)VF2 Primer (10(µM) | 0.75(µL)VF2 Primer (10(µM) |
1.25(µL)VF Primer (10(µM) | 0.75(µL)VF Primer (10(µM) |
0.2(µL) Pfu polymerase | 0.2(µL) Pfu polymerase |
11.8 Milli-Q H2O | 12.6 Milli-Q H2O |
- as control for each rxn used equal volume of mRBS maxiprep
Knight cont'd | Endy cont'd |
cycling conditions | cycling conditions |
950C for 15 minutes | 950C for 6 minutes |
*940C for 30 seconds | **950C for 30 seconds |
*560C for 30 seconds | **560C for 30 seconds |
*680C for 1 minutes | **700C for 1 minutes |
680C for 20 minutes | 700C for 10 minutes |
(*) were run 39 times
(**) were run 35 times
Made the following program (called COLONYY) Lid preheat 980C
- 980C for 15 minutes
- 980C for 30 seconds
- 560C for 30 seconds
- 68-700C gradient for 1 minute
- 68-700C gradient for 20 minute
- 40C indefinte
bold selections were cycled 39 times
Objective: Analyzed PCR products on 2.5% TAE Agarose gel.
lane | sample | loaded |
1 | 50bp ladder | 1(µL) ladder, 1(µL) dye, 4(µL) H20 |
2 | Knight control | 5(µL) sample, 1(µL)dye |
3 | Knight colony | 5(µL) sample, 1(µL)dye |
4 | Endy control | 5(µL) sample, 1(µL)dye |
5 | Endy colony | 5(µL) sample, 1(µL)dye |
6 | 50bp ladder | 1(µL) ladder, 1(µL) dye, 4(µL) H20 |
7 | lumazine(justin's) | 5(µL) sample, 1(µL)dye |
Repeat gel with template controls
lane | sample | loaded |
1 | 50bp ladder | 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20 |
2 | Endy Template | 5(µL) colony suspension, 1(µL)dye, 4(µL) H20 |
3 | Endy mRBS Control (PLR) | 5(µL) sample, 1(µL)dye |
4 | Endy mRBS-TetR colony(PCR) | 5(µL) sample, 1(µL)dye |
4 | mRBS template | 0.5(µL) sample, 1(µL)dye, 4(µL) H20 |
5 | Knight Template | 0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20 |
6 | Knight mRBS control | 5(µL) ladder, 1(µL) dye |
7 | Knight-mRBS-TetR colony | 5(µL) sample, 1(µL)dye |
1 | 1Kb ladder | 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20 |
- ran at 100V for 75 minutes
Aug 9/2010
AV