Team:Lethbridge/Notebook/Lab Work/August

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(Difference between revisions)
(→August 4/2010)
(→August)
Line 266: Line 266:
<td><b>results</b>
<td><b>results</b>
<tr><td>contents<td><b>&250&micro;L</b><td><b>150&micro;L</b>
<tr><td>contents<td><b>&250&micro;L</b><td><b>150&micro;L</b>
-
<tr><td>dt-pTet<td><b>:)</b><td>x
+
<tr><td>dt-pTet<td>good<td>x
<tr><td>- control<td>x<td>x
<tr><td>- control<td>x<td>x
-
<tr><td>mms6-pET-28a<td><b>:)</b><td><b>:)</b>
+
<tr><td>mms6-pET-28a<td>good<td>good
<tr><td>dt-pSBIC3<td>x<td>x
<tr><td>dt-pSBIC3<td>x<td>x
-
<tr><td>mRBS-TetR<td><b>:)</b><td><b>:)</b>
+
<tr><td>mRBS-TetR<td>good<td>good
-
<tr><td>pLacI-mRBS<td><b>:)</b><td><b>:)</b>
+
<tr><td>pLacI-mRBS<td>good<td>good
<tr><td>SRBS-TetR<td>x<td>x
<tr><td>SRBS-TetR<td>x<td>x
-
<tr><td>pBAD-SRBS<td><b>:)</b><td><b>:)</b>
+
<tr><td>pBAD-SRBS<td>good<td>good
-
<tr><td>+ contol<td><b>:)</b><td><b>:)</b>
+
<tr><td>+ contol<td>good<td>good
</table><br>
</table><br>
-
<font color ="red">"AUG 4" is not yet done</font>
 
==August 6/2010 Evening==
==August 6/2010 Evening==
Line 286: Line 285:
*Knight Protocol<br>
*Knight Protocol<br>
-
**place 20micro
+
**place 20(&micro;L) sterile H<sub>2</sub>O in 0.6mL sterile tube
 +
**with P10 pipette set to 3(&micro;L) dip tip into colony
 +
**place pipette tip into water and pipette up and down 20 times(this can be stored at 4<sup>0</sup>C for inoculation of overnight 5mL cultures)<br>
 +
 
 +
*Endy Protocol
 +
**place 50(&micro;L) sterile H<sub>2</sub>O in 0.6mL sterile tube
 +
**with PLO pipette (set 3(&micro;L)) dip sterile tip into colony
 +
**place pipette tip into water and pipette up and down 20 times
 +
 
 +
<table border ="3">
 +
<tr><td><b>Knight cont'd</b><td><b>Endy cont'd</b>
 +
<tr><td><b>setup 20(&micro;L) reaction</b><td><b>setup 20(&micro;L) reaction</b>
 +
<tr><td>1(&micro;L) colony suspension<td>2(&micro;L) colony suspension
 +
<tr><td>2(&micro;L) 10x p.fu (+Mg SO<sub>4</sub><td>2(&micro;L) 10x p.fu (+Mg SO<sub>4</sub>
 +
<tr><td>2(&micro;L) dNTP<td>2(&micro;L) dNTP
 +
<tr><td>1.25(&micro;L)VF2 Primer (10(&micro;M)<td>0.75(&micro;L)VF2 Primer (10(&micro;M)
 +
<tr><td>1.25(&micro;L)VF Primer (10(&micro;M)<td>0.75(&micro;L)VF Primer (10(&micro;M)
 +
<tr><td>0.2(&micro;L) Pfu polymerase<td>0.2(&micro;L) Pfu polymerase
 +
<tr><td>11.8 Milli-Q H<sub>2</sub>O<td>12.6 Milli-Q H<sub>2</sub>O
 +
</table><br>
 +
 
 +
*as control for each rxn used equal volume of mRBS maxiprep
 +
 
 +
<table border ="3">
 +
<tr><td><b>Knight cont'd</b><td><b>Endy cont'd</b>
 +
<tr><td><b>cycling conditions</b><td><b>cycling conditions</b>
 +
<tr><td>95<sup>0</sup>C for 15 minutes<td>95<sup>0</sup>C for 6 minutes
 +
<tr><td>*94<sup>0</sup>C for 30 seconds<td>**95<sup>0</sup>C for 30 seconds
 +
<tr><td>*56<sup>0</sup>C for 30 seconds<td>**56<sup>0</sup>C for 30 seconds
 +
<tr><td>*68<sup>0</sup>C for 1 minutes<td>**70<sup>0</sup>C for 1 minutes
 +
<tr><td>68<sup>0</sup>C for 20 minutes<td>70<sup>0</sup>C for 10 minutes
 +
</table><br>
 +
 
 +
(*) were run 39 times<br>
 +
(**) were run 35 times<br>
 +
 
 +
Made the following program (called COLONYY)
 +
Lid preheat 98<sup>0</sup>C
 +
*98<sup>0</sup>C for 15 minutes
 +
*<b>98<sup>0</sup>C for 30 seconds</b>
 +
*<b>56<sup>0</sup>C for 30 seconds</b>
 +
*<b>68-70<sup>0</sup>C gradient for 1 minute</b>
 +
*68-70<sup>0</sup>C gradient for 20 minute
 +
*4<sup>0</sup>C indefinte
 +
 
 +
<b>bold</b> selections were cycled 39 times<br>
 +
 
 +
<b> Objective:</b> Analyzed PCR products on 2.5% TAE Agarose gel.
 +
 
 +
<table border ="3">
 +
<tr><td><b>lane</b><td><b>sample</b><td><b>loaded</b>
 +
<tr><td>1<td>50bp ladder<td>1(&micro;L) ladder, 1(&micro;L) dye, 4(&micro;L) H<sub>2</sub>0
 +
<tr><td>2<td>Knight control<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>3<td>Knight colony<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>4<td>Endy control<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>5<td>Endy colony<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>6<td>50bp ladder<td>1(&micro;L) ladder, 1(&micro;L) dye, 4(&micro;L) H<sub>2</sub>0
 +
<tr><td>7<td>lumazine(justin's)<td>5(&micro;L) sample, 1(&micro;L)dye
 +
</table><br>
 +
 
 +
Repeat gel with template controls
 +
 
 +
<table border  ="3">
 +
<tr><td><b>lane</b><td><b>sample</b><td><b>loaded</b>
 +
<tr><td>1<td>50bp ladder<td>0.5(&micro;L) ladder, 2(&micro;L) dye, 9.5(&micro;L) H<sub>2</sub>0
 +
<tr><td>2<td>Endy Template<td>5(&micro;L) colony suspension, 1(&micro;L)dye, 4(&micro;L) H<sub>2</sub>0
 +
<tr><td>3<td>Endy mRBS Control (PLR)<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>4<td>Endy mRBS-TetR colony(PCR)<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>4<td>mRBS template<td>0.5(&micro;L) sample, 1(&micro;L)dye, 4(&micro;L) H<sub>2</sub>0
 +
<tr><td>5<td>Knight Template<td>0.25(&micro;L) colony suspension, 1(&micro;L)dye, 4(&micro;L) H<sub>2</sub>0
 +
<tr><td>6<td>Knight mRBS control<td>5(&micro;L) ladder, 1(&micro;L) dye
 +
<tr><td>7<td>Knight-mRBS-TetR colony<td>5(&micro;L) sample, 1(&micro;L)dye
 +
<tr><td>1<td>1Kb ladder<td>0.5(&micro;L) ladder, 2(&micro;L) dye, 9.5(&micro;L) H<sub>2</sub>0
 +
</table><br>
 +
 
 +
*ran at 100V for 75 minutes
 +
 
 +
==Aug 9/2010==
 +
 
 +
AV
 +
 
 +
<center><b> Objective:</b> To do Glycerol stocks/miniprep the following</center>

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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
ConstructpDNAbufferEnzymes
pBAD-sRBS/mRBSpBADRedEcoRI and SpeI
pBAD-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pBAD-sRBS/mRBSmRBSOrangeXbaI and EcoRII
sRBS/mRBS-TetRsRBSRedPstI and SpeI
sRBS/mRBS-TetRmRBSRedPstI and SpeI
sRBS/mRBS-TetRTetRTangoXbaI and PstI
TetR-dTTetRRedEcoRI and SpeI
TetR-dTdTOrangeXbaI and EcoRI
dT-pTetdTRedEcoRI and SpeI
dT-pTetpTetOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSpLacIRedEcoRI and SpeI
pLAcI-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSmRBSOrangeXbaI and EcoRI
Mms6-PET28(a)PET28(a)OrangeNotI
  • 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

ComponentVolume per tube (µL)Master Mix (x6)
MilliQ H2O41.8250.8
10x Pfu Buffer with MgSO4530
dNTPs16
Forward Primer0.53
Reverse Primer0.53
Template DNA1-
Pfu DNA polymerase0.2-
Total50292.8
  • 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:

LaneSampleComponents (µL)
11 kb ladder2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O
2ECFP2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
3EYFP2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
4Lumazine2 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:

RestrictionMilliQ H2O (µL)Buffer Orange (µL)pDNA (µL)Enzyme (µL)
pSB1C379.2510100.25 EcoRI + 0.25 PstI + 0.25 DpnI
pSB1C3 control801010-
dT79.5010100.25 EcoRI + 0.25 PstI
dT control801010-
  • 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.

LaneContentsResult
11kb ladder
2pTet (XbaI, EcoRI)good
3pTet (orange buffer)---
4dT (XbaI, EcoR)no good
5dT (orange buffer)---
6mRBS (SpeI, PstI) good
7mRBS (red buffer)---
8sRBS (SpeI, PstI) good
9sRBS (red buffer)---
10mRBS (XbaI, EcoRI) good
11mRBS (orange buffer)---
12sRBS (XbaI, EcoRl) good
13sRBS (orange buffer)---
14pet28(a) good
15100 bp ladder
16PSB1C3 not good
17PSB1C3 restriction digest---

LaneContentsResult
1TetR (EcorI, SpeI) good
2TetR (red buffer)---
3pLacI (EcoRI, SpeI) good
4pLacI (red buffer)---
5Mms6can not tell
6Mms6 control---
7TetR (Xbal, PstI) good
8TetR (tango buffer)---
9pBAD (EcoRI, SpeI)good
10pBAD (red buffer)---
11dT (EcoRI, SpeI)good
12dT (red buffer)---
13pLacI (2)?
14dT control not good
15dT restriction
16100 bp ladder
17dT PCR product good
18Mms6 PCR product good
19pBAD PCR product good
20pLacI 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

Component1X(µL)Master Mix(x16)(µL)
Milli-Q H2O41.8668.6
10x Pfu Buffer with MgSO4580
dNTPs116
Forward Primer0.58
Reverse Primers0.58
Template DNA116
Pfu polymerase0.23.2

2.5% agarose gel(1x TAE)

lanecontentsSuccessful Ligation ?
150bp ladder---
2dt pSBIC3---
3dt pTetx
4dt control---
5sRBS-TetRx
6mRBS-TetR?
7TetR control---
8pLacI-mRBSx
9pLacI-sRBS?
10pLacI control---
11Mms6 pET-28ano band
12Mms6 control---
13pBad-SRBSx
14pBad-mRBSx
15pBad 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&250µL150µL
dt-pTetgoodx
- controlxx
mms6-pET-28agoodgood
dt-pSBIC3xx
mRBS-TetRgoodgood
pLacI-mRBSgoodgood
SRBS-TetRxx
pBAD-SRBSgoodgood
+ contolgoodgood


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'dEndy cont'd
setup 20(µL) reactionsetup 20(µL) reaction
1(µL) colony suspension2(µL) colony suspension
2(µL) 10x p.fu (+Mg SO42(µL) 10x p.fu (+Mg SO4
2(µL) dNTP2(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 polymerase0.2(µL) Pfu polymerase
11.8 Milli-Q H2O12.6 Milli-Q H2O

  • as control for each rxn used equal volume of mRBS maxiprep
Knight cont'dEndy cont'd
cycling conditionscycling conditions
950C for 15 minutes950C 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 minutes700C 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.

lanesampleloaded
150bp ladder1(µL) ladder, 1(µL) dye, 4(µL) H20
2Knight control5(µL) sample, 1(µL)dye
3Knight colony5(µL) sample, 1(µL)dye
4Endy control5(µL) sample, 1(µL)dye
5Endy colony5(µL) sample, 1(µL)dye
650bp ladder1(µL) ladder, 1(µL) dye, 4(µL) H20
7lumazine(justin's)5(µL) sample, 1(µL)dye

Repeat gel with template controls

lanesampleloaded
150bp ladder0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20
2Endy Template5(µL) colony suspension, 1(µL)dye, 4(µL) H20
3Endy mRBS Control (PLR)5(µL) sample, 1(µL)dye
4Endy mRBS-TetR colony(PCR)5(µL) sample, 1(µL)dye
4mRBS template0.5(µL) sample, 1(µL)dye, 4(µL) H20
5Knight Template0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20
6Knight mRBS control5(µL) ladder, 1(µL) dye
7Knight-mRBS-TetR colony5(µL) sample, 1(µL)dye
11Kb ladder0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20

  • ran at 100V for 75 minutes

Aug 9/2010

AV

Objective: To do Glycerol stocks/miniprep the following
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