Team:Lethbridge/Notebook

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Revision as of 19:01, 21 October 2009

September

September 3

Roxanne, Ashley, Kirsten

Made 1.5L agar LB media according to lab protocol. Roxanne is autoclaving tomorrow.

Pick 3 lumazine-dT colonies from glycerol stocks to grow overnight

Restriction Digest

Reaction 1

	Ribo-cheZ-dT	GFP-dT	sRBS-mmS6	Mr. Gene mmS6
milliQ water	5 µL	5 µL	5 µL	5 µL
Buffer tango	3 µL	3 µL	3 µL	3 µL
DNA	20 µL	20 µL	20 µL	20 µL
XbaI	1 µL	1 µL	1 µL	1 µL
PstI	1 µL	1 µL	1 µL	1 µL
total	30 µL	30 µL	30 µL	30 µL

Reaction 2

	Ribo-cheZ-dT	GFP-dT	sRBS-mmS6	Mr. Gene mmS6
milliQ water	3 µL	3 µL	3 µL	3 µL
Buffer tango	1 µL	1 µL	1 µL	1 µL
DNA	5 µL	5 µL	5 µL	5 µL
EcoRI	1 µL	1 µL	1 µL	1 µL
total	10 µL	10 µL	10 µL	10 µL

Into HWB at 37°C for 2 hours

Gel Electrophoresis (concentration):

Lane

1kb ladder
ribo-cheZ-dT (EcoRI)
GFP-dT (EcoRI)
sRBS-mmS6-dT (EcoRI)
NEYFP (XbaI,PstI)
NEYFP (ecoRI, speI)
NCFP (ecoRI, speI)
NECFP (xbaI,pstI)
CCFP(ecoRI,speI)
CECFP(ecoRI,speI)
CCFP (notI,pstI)
NEYFP (ecoRI,speI)
CECFP (xbaI)
CEYFP(xbaI,pstI)
NCFP (xbaI,pstI)
CEYFP(xbaI)
pLacI
pStrong
1kb ladder

5µL sample, 1 µL loading dye (6x) 6 µL ladder

Ran at 100V for 1 hour

Concentrations good!

Alix

Gel extraction of:

sRBS-mmS6-dT

riboswitch-cheZ-dT

GFP-dT

mmS6

Following Qiagen benchtop protocol

September 4

Ashley

Ligation of:

C-term-dT +C-CFP #2
C-term-dT +C-ECFP #2
C-term-dT + C-EYFP #2
pSB1A3-2+C-CFP#1
pSB1A3-2+C-ECFP#1
pSB1A3-2+C-EYFP#1
mRBS-N-term +N-CFP#1
mRBS-N-term +N-EYFP#1
mRBS-N-term +N-ECFP#1
pSB1A3-1+N-CFP#2
pSB1A3-1+N-ECFP#2
pSB1A3-1+N-EYFP#2

Reaction

	Control	All reactions
milliQ water	7.5 µL	4.5 µL
10x buffer T4	1 µL	1 µL
Vector	1 µL	1 µL
Insert	0 µL	3 µL
T4 DNA ligase	0.5 µL	0.5 µL
total	10 µL	10 µL

Restriction Digest

	Control (no enzyme)	Lumazine-dT (1)
milliQ water	2.4 µL	0.4 µL
10x buffer Tango	1.6 µL	1.6 µL
DNA	16 µL	16 µL
XbaI	0 µL	1 µL
PstI	0 µL	1 µL
total	20 µL	20 µL

September 8

Ashley

Made 1.5L LB agar according to protocol

Made 300mL LB broth according to protocol

Gel Electrophoresis (for extraction)

5 µL ladder

5 µL sample, 1 µL dye

Lane

1 kb ladder
empty^
empty
lumazine-dT (XbaI, PstI) 600bp

^there was a control (no enzyme) but not enough DNA to load

Ran at 1000V for one hour

Gel extraction according to Qiagen benchtop protocol

Sept 10

Ashley, Kirsten

Ran 1% agarose

Lane

1 kb ladder
C-term-dT + C-EYFP #2
pSB1A3-1+N-CFP#2
pSB1A3-1+N-ECFP#2
lumazine+sRBS
pSB1A3-2+C-EYFP#1
pSB1A3-1+N-EYFP#2
mRBS-N-term +N-CFP#1
mmS6+pSB1A3-1
GFP-dT
mmS6-dT
pSB1A3-2+C-ECFP#1
mRBS-N-term +N-ECFP#1
mRBS-N-term +N-EYFP#1
C-term-dT +C-CFP #2
C-term-dT +C-ECFP #2
pSB1A3-2+C-CFP#1

3 µL sample + 0.5 µL dye

6 µL 1 kb ladder

Mackenzie

Transformation into DH5α

sRBS-lumazine-dT (km)
mmS6-dT (km)
pSB1A3+mmS6
mRBS-N-term +N-ECFP#1
mRBS-N-term +N-EYFP#1
C-term-dT +C-ECFP #2
C-term-dT + C-EYFP #2
pSB1A3-2+C-ECFP#1
pSB1A3-2+C-EYFP#1

The following were not transformed due to lack of competent cells:

pSB1A3-1+N-ECFP#2
pSB1A3-1+N-EYFP#2
GFP-dT

September 14

Ashley

PCR of EYFP, CFP and ECFP

See Aug 6 for volumes

CFP(1)=prefix primer and antisense C-term suffix

CFP(2)=N-term prefix and antisense suffix

EYFP(1)=prefix primer and antisense C-term suffix

EYFP(2)=N-term prefix and antisense suffix

ECFP(1)=prefix primer and antisense C-term suffix

ECFP(2)=N-term prefix and antisense suffix

Cycle: tail67

Sept 16

Ashley

Miniprep of mmS6-dT 1,2 and 3. Used Qiagen benchtop protocol and kit. Eluted with 50 µL buffer EB. Put into -20°C freezer orange tray.

PCR Purification of restriction digest of PCR products.

September 24

Ashley, Kirsten

1% gel electrophoresis 6uL ladder (1kb) 5uL sample +1 uL 6x loading dye 100V, 40min

Lane

1kb Ladder
C-CFP-1
C-ECFP-1
N-EYFP-2
N-CFP-2
N-ECFP-2
C-EYFP-1
CFP-1 Purified PCR
ECFP-1 Purified PCR
EYFP-2 Purified PCR
CFP-2 Purified PCR
ECFP-1 Purified PCR
EYFP-1 Purified PCR
C-CFP(1)
ECFP(1)
C-EYFP(1)
N-CFP(2)
N-EYCFP(2)
N-EYFP(2)
1KB LADDER

Gel was run to check for DNAses, found that DPN-1 had DNAses.

Sept 25

1% agarose gel 100V, 40min

Lane:

1kb ladder
mms6(EcoRI,SpeI)
pLacI (SpeI, PstI)
Lumazine-dT

Only pLacI success, gell extracted and eluted with 50uL buffer EB.

Miniprep of:

3x mmS6-dT
3x pSB1A3-mmS6
2x cheZ-dT
1x riboswitch-cheZ-dT
2x GFP-dT
3x C-EYFP-dT

Restricted all of the above with EcoRI

8uL DNA
1uL 10x buffer tango
1uL EcoRI

for 1 hour.

Restriction of fusion proteins with either EcoRI,SpeI or XbaI,PstI

Heat inactivated a 60C for 10min

Ran on a gel (1% agarose)100V,40min

Lane:

1kb ladder
pSB-mmS6 #1
pSB-mmS6 #2
pSB-mmS6 #3
mms6-dT #1
mms6-dT #2
mms6-dT #3
EYFP C-term-dT #1
EYFP C-term-dT #2
EYFP C-term-dT #3
GFP-dT#1
GFP-dT#2
Riboswitch-cheZ-dT
cheZ-dT#1
cheZ-dT#2
C-CFP (EcoRI,SpeI)
C-CFP (XbaI,PstI)
C-ECFP (EcoRI,SpeI)
C-ECFP (XbaI,PstI)
1kb laddder

Restriction of Fusion proteins with Dpn1
Heat inactivated a 60C for 10min

Lane:

1kB ladder
N-ECFP
N-ECFP
N-EYFP
E-EYFP
N-CFP
N-CFP
C-EYFP
C-EYFP
1kB ladder

Restriction of Lumazine-dT

30uL DNA
4uL 10x buffer
3uL EcoRI
3uL SpeI

Setup Liquid culture of the Mr. Gene mmS6 x3

September 28

Ashley

1% agarose gel

5uL DNA+1uL 6x loading dye
6uL ladder (1kb)

Lane:

1kb ladder
N-ECFP (XbaI,PstI)
N-ECFP (EcoRI, SpeI)
N-EYFP(XbaI,PstI)
N-EYFP(EcoRI, SpeI)
N-CFP(XbaI,PstI)
N-CFP(EcoRI, SpeI)
C-EYFP(XbaI,PstI)
C-EYFP(EcoRI, SpeI)
1kb ladder

Ran at 100V for 40 min

October

October 1

Roxanne Gel for gel extraction

Restriction Ribo-Chez-dT

2x CheZ-dT
2x GFP-dT
3x C-EYFP-dT

Ligation

C-ECFP-dT
C-CFP-dT
C-EYFP-dT
mRBS-N-ECFP
mRBS-N-EYFP
pSB-C-EFP
pSB-C-EYFP
pSB-C-CFP
pSB-N-ECFP
pSB-N-EYFP

Aliquoted DH5α cells

Ashley

Tube	Tube + Gel	Gel
1.1226g	1.4931g	0.2671g
1.012g	1.2302g	0.1290g

Eluted with 50uL buffer EB

Ligation

	1:3	2:6
lum-dT	3uL	6uL
SRBS	1uL	2uL
Ligase Buffer	1uL	2uL
Water	4.5uL	9uL
Ligase	0.5uL	1uL

Oct 2

Pick Colonies:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs

Gel Extraction:

Riboswitch-cheZ-dT
2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
3x C-EYFP-dT
2x GFP-dT

Liquid Culture:

Riboswitch-cheZ-dT
2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
3x C-EYFP-dT
2x GFP-dT

Miniprep:

Mr. Gene mms6

Sat Oct 3

Miniprep:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs
sRBS + Lumazine-dT

Restriction:

Analytic and Prep:

mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs

Analytic only:

Mr. Gene mms6

Prep only:

pBAD-TetR

Oct 5

Gel extraction:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs

Ligation:

pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT
pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term

Transformation:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Sequencing:

sRBS + Lumazine-dT
mRBS + N-term FPs
C-term FPs + dT
pSB + N-term FPs
pSB + N-term FPs
Mr. Gene mms6

Tues Oct 6

Pick Colonies:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Wed Oct 7

Miniprep:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + C-term ECFP
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Analytic Restriction:

pLac + sRBS-Lumazine-dT
pBad-TetR + C-term FPs
pBad-TetR + N-term FPs
pTetR + N-term ECFP
pStrong + Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Prep Restriction:

Riboswitch-GFP-dT

Thurs Oct 8

Gel Extraction:

Riboswitch-GFP-dT

Ligation:

pStrong + Riboswitch-GFP-dT

Transformation:

pStrong + Riboswitch-GFP-dT

Sequencing:

pLac-sRBS-Lumazine-dT
pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
pStrong-Riboswitch-cheZ-dT
Riboswitch-GFP-dT

Maxiprep:

pLac-sRBS-Lumazine-dT
pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
pStrong-Riboswitch-GFP-dT

Restriction:

pLac-sRBS-Lumazine-

Analytic and Prep:

pBad-TetR-C-term FPs
pBad-TetR-N-term FPs
pTetR-N-term ECFP
C-term CFP (has pTetR)

Analytic Only:

pStrong-Riboswitch-GFP-dT

Fri Oct 9 Overexpression pLac + sRBS-Lumazine-dT Gel Extraction pLac-sRBS-Lumazine-dT pBad-TetR-C-term FPs pBad-TetR-N-term FPs pTetR-N-term ECFP C-term CFP (has pTetR) Motility Media

Oct 12

Ligation:

pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Transformation:

pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Plate Testing:

pStrong-Riboswitch-GFP-dT

Motility Test:

pStrong + Riboswitch-cheZ-dT

Oct 13

Pick Colonies

pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Analysis of Plate and Motility Tests

Wed Oct 14

Miniprep:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Restriction:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Thurs Oct 15

Gel Extraction:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Ligation:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Transformation:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Expression Testing:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Fri Oct 16

Pick Colonies:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Sat Oct 17

Maxiprep:

pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

@@ Line 300: / Line 300: @@
-== August ==
-===Aug 4===
-'''Roxanne and Kirsten'''
-DNA purification of the 2 pSB1A3 plasmids, dT and pTet using the gel extraction kit
-Ran gel to check concentrations
-*pTet 25ng/µL
-*pSB1A3-2
-*pSB1A3-1
-*dT 180 ng/µL, 3318bp
-*N-term 25 ng/µL, 507bp
-*Lumazine 120 ng/µL, 861bp
-*c-term 25 ng/µL, 55bp
-*EYFP 878bp
-Ligations of:
-pTet-EYFP
-Lumazine-dT
-c-term-dT
-Calculations:
-EYFP: 3x(878bp [EYFP]/2211bp [pTet])x25ng
-=29.78ng
-=4µL EYFP:1 µL pTet
-µL Lumazine: 1 µL dT
-µL c-term:1 µL dt
-{|
-|
-|'''pTet-EYFP'''
-|''Lumazine-dT''
-|c-term-dT
-|-
-|milliQ water
-|'''3.5 µL'''
-|''6.5 µL''
-|6.5 µL
-|-
-|10x buffer
-|'''1 µL'''
-|''1 µL''
-|1 µL
-|-
-|insert
-|'''4 µL'''
-|''1 µL''
-|1 µL
-|-
-|Vector
-|'''1 µL'''
-|''1 µL''
-|1 µL
-|-
-|Ligase
-|'''0.5 µL'''
-|''0.5 µL''
-|0.5 µL
-|}
-'''Megan/Mackenzie'''
-Gel extraction pSBIA3-1 and 2 (plasmid for biobrick parts)
-Transformation: Tranformed to DH5alpha pBAD = promoter TetR= inverter. Added 2 micro liters of each ligation to DH5alpha. See July 14th Protocol
-Restrictions of pBAD (SpeI/PstI), TetR (XbaI/PstI), PLacI (SpeI, PstI), sRBS (SpeI/ PstI), mRBS (SpeI/PstI). Used PCR tubes. Each tube contains: 5 micro liters MillQ water, 3 micro liters 10x Tango Buffer, 1 micro liter enzyme 1, 1 micro liter enzyme 2, 20 micro liters of DNA. Put all tubes in thermal controller at 37 degrees.
-Roxanne,
-Gel Extraction of pBAD, TetR, pLacI, sRBS, mRBS
-Lengths:
-pBAD:2287 bp
-TetR: 902 bp
-pLacI: 2279
-sRBS: 2092 bp
-mRBS: 2092 bp
-Lane:
-#Ladder
-#TetR
-#pLacI
-#sRBS
-#mRBS
-#pBAD
-[[Image:Aug4.png|250px]]
-===Aug 5th===
-'''Kirsten and Fan'''
-Gel extraction of TetR, pLacI, sRBS, mRBS, pBAD according to Qiagen protocol.
-Ran analytic gel
-µL Dye + 5µL DNA
-µL Ladder
-Lane:
-#Ladder
-#TetR
-#pLacI
-#sRBS
-#mRBS
-#pBAD
-#pSB1A3-1
-#pSB1A3-2
-[[Image:Aug5.JPG|250px]]
-tetR concentration: 25ng/µL
-everything else: 100ngµL
-<html>
-<head>
-<!--%%%%%%%%%%%% QuickMenu Styles [Keep in head for full validation!] %%%%%%%%%%%-->
-<style type="text/css">
-/*!!!!!!!!!!! QuickMenu Core CSS [Do Not Modify!] !!!!!!!!!!!!!*/
-.qmmc .qmdivider{display:block;font-size:1px;border-width:0px;border-style:solid;position:relative;z-index:1;}.qmmc .qmdividery{float:left;width:0px;}.qmmc .qmtitle{display:block;cursor:default;white-space:nowrap;position:relative;z-index:1;}.qmclear {font-size:1px;height:0px;width:0px;clear:left;line-height:0px;display:block;float:none !important;}.qmmc {position:relative;zoom:1;z-index:10;}.qmmc a, .qmmc li {float:left;display:block;white-space:nowrap;position:relative;z-index:1;}.qmmc div a, .qmmc ul a, .qmmc ul li {float:none;}.qmsh div a {float:left;}.qmmc div{visibility:hidden;position:absolute;}.qmmc .qmcbox{cursor:default;display:block;position:relative;z-index:1;}.qmmc .qmcbox a{display:inline;}.qmmc .qmcbox div{float:none;position:static;visibility:inherit;left:auto;}.qmmc li {z-index:auto;}.qmmc ul {left:-10000px;position:absolute;z-index:10;}.qmmc, .qmmc ul {list-style:none;padding:0px;margin:0px;}.qmmc li a {float:none;}.qmmc li:hover>ul{left:auto;}#qm0 ul {top:100%;}#qm0 ul li:hover>ul{top:0px;left:100%;}
-/*!!!!!!!!!!! QuickMenu Styles [Please Modify!] !!!!!!!!!!!*/
-	/* QuickMenu 0 */
-	/*"""""""" (MAIN) Container""""""""*/
-	#qm0
-	{
-		width:auto;
-		background-color:transparent;
-	}
-	/*"""""""" (MAIN) Items""""""""*/
-	#qm0 a
-	{
-		padding:5px 60px 3px 0px;
-		margin:0px 5px 0px 0px;
-		color:#FFD600;
-		font-family:Arial;
-		font-size:12px;
-		text-decoration:none;
-		font-weight:bold;
-		border-width:0px 0px 8px 0px;
-		border-style:solid;
-		border-color:#FFD600;
-	}
-	/*"""""""" (MAIN) Hover State""""""""*/
-	#qm0 a:hover
-	{
-		color:#3C13AF;
-		border-color:#FFAD00;
-	}
-	/*"""""""" (MAIN) Hover State - (duplicated for pure CSS)""""""""*/
-	#qm0 li:hover>a
-	{
-		color:#3C13AF;
-		border-color:#FFAD00;
-	}
-	/*"""""""" (MAIN) Parent Items""""""""*/
-	#qm0 .qmparent
-	{
-		background-repeat:no-repeat;
-		background-position:95% 50%;
-	}
-	/*"""""""" (MAIN) Active State""""""""*/
-	body #qm0 .qmactive, body #qm0 .qmactive:hover
-	{
-		color:#3C13AF;
-		text-decoration:none;
-		border-color:#FFAD00;
-	}
-	/*"""""""" (SUB) Container""""""""*/
-	#qm0 div, #qm0 ul
-	{
-		padding:10px 5px 10px 5px;
-		background-color:#EEEEEE;
-		border-width:1px;
-		border-style:solid;
-		border-color:#3C13AF;
-	}
-	/*"""""""" (SUB) Items""""""""*/
-	#qm0 div a, #qm0 ul a
-	{
-		padding:3px 5px 3px 5px;
-		background-color:transparent;
-		color:#230672;
-		font-size:11px;
-		font-weight:normal;
-		border-width:0px;
-		border-style:none;
-		border-color:#230672;
-	}
-	/*"""""""" (SUB) Hover State""""""""*/
-	#qm0 div a:hover
-	{
-		background-color:#476DD5;
-		color:#FFFFFF;
-		text-decoration:none;
-	}
-	/*"""""""" (SUB) Hover State - (duplicated for pure CSS)""""""""*/
-	#qm0 ul li:hover>a
-	{
-		background-color:#476DD5;
-		color:#FFFFFF;
-		text-decoration:none;
-	}
-	/*"""""""" (SUB) Parent Items""""""""*/
-	#qm0 div .qmparent, #qm0 ul .qmparent
-	{
-		background-image:url(qmimages/arrow_0.gif);
-	}
-	/*"""""""" (SUB) Active State""""""""*/
-	body #qm0 div .qmactive, body #qm0 div .qmactive:hover
-	{
-		background-color:#476DD5;
-		background-image:url(qmimages/arrow_1.gif);
-		color:#FFFFFF;
-	}
-	/*"""""""" Individual Titles""""""""*/
-	#qm0 .qmtitle
-	{
-		margin:2px 5px 5px 5px;
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-</head>
-<body>
-<ul id="qm0" class="qmmc">
-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
-	<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
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-===Aug 6===
-'''Roxanne'''
-Set up the following ligations
-pSB1A3-2+n-term tag and
-pSB1A3-1+c-term tag
-*6.5µL milliQ water
-*1 µL 10x T4 buffer
-*1 µL insert
-*1 µL vector
-*0.5 µL T4 ligase
-pSB1A3+lumazine
-*5.5µL milliQ water
-*1 µL 10x T4 buffer
-*2 µL insert
-*1 µL vector
-*0.5 µL T4 ligase
-pBAD+tetR inverter
-*2.5µL milliQ water
-*1 µL 10x T4 buffer
-*5 µL insert
-*1 µL vector
-*0.5 µL T4 ligase
-mRBS+n-term tag
-*6.5µL milliQ water
-*1 µL 10x T4 buffer
-*1 µL insert
-*1 µL vector
-*0.5 µL T4 ligase
-Colony PCR of pTet-EYFP, lumazine-dT, c-term-dT
-Picked 5 colonies of each construct, followed protocol of July 23.
-E1-E5=EYFP colonies 1-5
-E+=EYFP (no pTet)
-L1-L5=lumazine-dT colonies 1-5
-L+= lumazine (no dT)
-C1-C5= c-term-dT colonies 1-5
-C+= c-terminal (no dT)
--=no plasmid
-'''Lisza'''
-Mixed 10mM primers for: antisense suffix primer and FP N-term fusion primer
-'''Ashley, Mackenzie, Lisza'''
-Analytical gel of colony PCR’s (from earlier)
-Volumes:
-*10 µL DNA
-*2 µL 6x loading
-*10 µL ladder
-Lane
-#1 kb ladder
-#–ve control
-#L1
-#L2
-#L3
-#L4
-#L5
-#L+
-#E1
-#E2
-#E3
-#E4
-#E5
-#E+
-#C1
-#C2
-#C3
-#C4
-#C5
-#C+
-[[Image:Aug_6.jpg|250px]]
-Transformation of mRBS+n-term, pBAD+tetR, pSB1A3+lumazine, pSB1A3+c-term, pSB1A3+n-term
-Negative control: water+25 µL DNA
-Transformed according to iGEM protocol.  Plated on ampicillin plates at 100µL and 400 µL volumes and incubated overnight.
-PCR of EYFP and CFP according to protocol of July 30th.
-===Aug 7===
-The transformations seemed to have all worked, although there are no colonies on plates marked pSBB1A3+N-term and several on the negative control plate.  Speculation: tubes were switched or improper labeling.
-Solution: Colony PCR
-Lane:
-#Ladder (1kb)
-#Negative
-#TetR
-#pBAD+tetR colony 1
-#pBAD+tetR colony 2
-#pBAD+tetR colony 3
-#mRBS
-#mRBS+nterm  colony 1
-#mRBS+nterm  colony 2
-#mRBS+nterm  colony 3
-#pSB1A3
-#c-term colony 1
-#c-term colony 2
-#c-term colony 3
-#N-term colony 1
-#N-term colony 2
-#N-term colony 3
-#Lumazine colony 1
-#Lumazine colony 2
-#Lumazine colony 3
-Set up restriction digest of the preperative PCR of EYFP (c-term) and CFP(c-term) fusion proteins
-DPNI, XbaI,PstI
-DPNI, EcoRI, speI for the n-term proteins
-Picked colonies of the transformed cells for pBAD-TetR, pSB1A3 (n-term), pSB1A3 (c-term), pSB1A3 (lumazine) and mRBS-Nterm
-x in ampicillin 5mL tubes.
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-</head>
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-<ul id="qm0" class="qmmc">
-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
-	<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
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-</html>
-===Aug 11===
-'''Roxanne'''
-Restricted all the minipreps with EcoRI to check the size of the plasmids prior to sending away for sequencing (10µL rxns)
-Restricted:
-*pSB-(n-term)  w/ SpeI, PstI
-*pSB-(c-term) w/ EcoRI, XbaI
-in 30 µL rxns to prepare for fusion to CFP of EYFP
-Setting up ligations of:
-*pSB1A3 + EYFP (c-term)
-*pSB1A3 + CFP (c-term)
-*pSB1A3 + EYFP (n-term)
-*pSB1A3 + CFP (n-term)
-mmS6 gene was transformed into DH5α by Megan and Fan, set up a few cultures for minipreps.
-===Aug 13===
-Set up restrictions of :
-*mmS6 (EcoRI, SpeI)
-*n-term CFP (EcoRI, SpeI)
-*CheZ (EcoRI, SpeI)
-*mRBS-N-term (SpeI, PstI)
-*c-term-dt (EcoRI, XbaI)
-Gel Extractions of:
-*mms6
-*cheZ
-Using Qiagen bench top protocol
-Set up ligations of:
-*pSB1A3-1 + mmS6
-*pSB1A3-1 + riboswitch
-*pSB1A3-1 + n-term CFP
-*pSB1A3-1 + n-term EYFP
-*pSB1A3-2 + c-term CFP
-*pSB1A3-2 + c-term EYFP
-*mRBS + N-term CFP
-*mRBS + N-term EYFP
-*CheZ+dT
-*mmS6 + dT
-Ran a concentration gel of:
-*c-term CFP
-*c-term EYFP
-*N-term CFP
-*N-term EYFP
-*c-term-dT
-*mRBS-N-term
-Concentrations:
-*Riboswitch= 100ng/µL
-*pSB1A3-1= 100ng/µL
-*pSB1A3-2= 100ng/µL
-*dT= 180ng/µL
-Ran 1% agarose gel (with 2 wells taped together)
-Lane:
-#5µL of 1kb ladder
-#20µL CheZ, 4µL dy
-Ran at 110V for 30min
-Gel extraction and purification of CheZ and mms6, mmS6 split into two tubes. Following Qiagen kit purification benchtop protocol.
-Concentration Gel (1% agarose)
-Lane:
-#1 kb ladder (6µL)
-#N-term EYFP
-#C-N
-#Y-N
-#4
-#1
-#mmS6
-#C-term dT
-#mRBS+n-term
-[[Image:Concentration_Gel_Aug_13.jpg|250px]]
-Only 9/18 transformations worked
-Success:
-*mmS6 in pSB1A3
-*mmS6 in dT
-*riboswitch in pSB1A3
-*CheZ in dT
-*CFP (no promoter)
-*GFP gene
-*pSB1A3 + n-term EFYP (1)
-*pSB1A3 +n-term EYFP (2)
-*fused c-EYFP
-Added more ligase and will re-transform the following:
-*Fused NEYFP (1)
-*Fused NEYFP (2)
-*Fused NCFP (1)
-*Fused NCFP (2)
-*Fused CEYFP
-*Fused CCFP
-*pSB1A3 +N-CFP (1)
-*pSB1A3 +N-CFP (2)
-*pSB1A3 +C-CFP (1)
-*pSB1A3 +C-EYFP (1)
-Transformed all of the above.  Picked colonies of successes.
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-===Aug 15th===
-The transformations didn’t work! Restart
-Picked 3 colonies of cheZ-dT
-of mmS6+dT (for mini and maxiprep)
-of fused C-EYFP
-of pSB1A3-mmS6
-of riboswitch in pSB1A3
-of GFP gene
-of CFP (no promoter)
-of the riboswitch and all of the N-EYFP in pSB1A3 turned red suggesting that the RFP reporter was religated in.
-Will miniprep the “good” cells and restrict with EcoRI to check for size
-Glycerol stock.
-===Aug 18===
-'''Megan and Ashley'''
-Pelleted down cells (small samples and large samples) picked from transformations to prep for mini-preps.
-Add 750 x2 micro liters of your small sample solution to centrifuge tube, Add 50 mL of large sample to large centrifuge tubes. Balanced the Centrifuge and ran for 2 minutes at 13000 rpm. Dump out Supernatant/ leave pellet. Add more sample, centrifuge, and remove more supernatant. Add remainder of sample, centrifuge, remove supernatant. Left samples in -20 degree freezer.
-===Aug 19===
-'''Kirsten and Mackenzie'''
-Maxiprep of fused C-EYFP and mmS6-dT
-See protocol for July 13th.
-===Aug 20===
-'''Fan and Mackenzie'''
-Miniprep for:
-*cheZ-dT
-*mmS6-dT
-*PSB1A3-mmS6
-*GFP
-*CFP no promo
-*Fused C-EYFP
-*Riboswitch (2,4,7,8,9)
-Restriction digest with EcoRI
-*10µL DNA
-*2 µL 10x buffer tango
-*4 µL EcoRI
-*Total volume = 20 µL
-°C for 1 hour.
-Need to run agarose for all restriction digested samples and DNA samples
-'''Roxanne, Mackenzie'''
-Running a gel of restricted minipreps
-Checking mini prep concentration with UV spec
-Lane
-#1 kb ladder
-#cheZ-dT 1 (78 µg/mL)
-#cheZ-dT 2 (80 µg/mL)
-#cheZ-dT 3 (106 µg/mL)
-#mmS6-dT 1 (82 µg/mL)
-#mmS6-dT 2(91 µg/mL)
-#mmS6-dT 3 (144 µg/mL))
-#pSB-mmS6 1 (125 µg/mL)
-#pSB-mmS6 2(88 µg/mL)
-#pSB-mmS6 3(135 µg/mL)
-#GFP 1(-------)
-#GFP 2 (108 µg/mL))
-#GFP 3(98 µg/mL)
-#CFP no promo 1(----------)
-#CFP no promo 2 (134 µg/mL)
-#Fused C-EYFP 1 (49 µg/mL)
-#Fused C-EYFP 2 (174 µg/mL)
-#Fused-CEYFP 3 (75 µg/mL)
-#1 kb ladder)
-[[Image:Aug_20,_big_gel.jpg|250px]]
-Lane
-#6 µL 1 kb ladder
-#6 µL pSB1A3
-#Riboswitch 2 (152 µg/mL)
-#Riboswitch 4 (81 µg/mL)
-#Riboswitch 7 (68 µg/mL)
-#Riboswitch 8 (112 µg/mL)
-#Riboswitch 9 (88 µg/mL)
-#6 µL 1 kb ladder
-[[Image:Aug_20,_little_gel.jpg|250px]]
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-</head>
-<body>
-<ul id="qm0" class="qmmc">
-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
-	<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
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-</body>
-</html>
-===Aug 25===
-'''Roxanne'''
-Set up restrictions
-,4,7 are c-term
-,5,8 are n-term
-,2=EYFP
-,5=CFP
-,8=ECFP
-*Fusion 1,4,7 restrict with DPN1, EcoRI, SpeI
-*Fusion 1,4,7 restrict with DPN1, XbaI, PstI
-*Fusion 2,5,8 restrict with DPN1, EcoRI, SpeI
-*Fusion 2,5,8, restrict with DPN1, XbaI, PstI
-*Riboswitch restrict with SpeI, PstI
-*CheZ-dT restrict with XbaI, PstI
-*GFP restrict with EcoRI, SpeI
-*4 µL milliQ water
-*2 µL 10x Tango buffer
-*12 µL DNA
-*1 µL E1
-*1 µL E1
-Set up maxipreps for:
-*Lumazine-dT
-*mmS6-dT
-Ligations:
-*Fusion 1 + (c-term)-dT
-*Fusion 4 + (c-term)-dT
-*Fusion 7 + (c-term)-dT
-*Fusion 1 + pSB1A3-2
-*Fusion 4 + pSB1A3-2
-*Fusion 7 + pSB1A3-2
-*Fusion 2 + mRBS (n-term)
-*Fusion 5 + mRBS (n-term)
-*Fusion 8 + mRBS (n-term)
-*Fusion 2 +pSB1A3-1
-*Fusion 5 +pSB1A3-1
-*Fusion 8 +pSB1A3-1
-*Riboswitch +CheZ-dT
-*GFP + dT
-*sRBS + lumazine-dT
-*sRBS + mmS6-dT
-===Aug 26===
-'''Lisza'''
-Deactivated to reactions @ 65° for 15 min
-'''Roxanne'''
-Set up liquid cultures of:
-*mmS6 in pSB1A3
-*mmS6-dT
-*lumazine in pSB1A3
-*lumazin-dT
-*c-term in pSB1A3
-*c-term-dT
-*n-term in pSB1A3
-*mRBS-(n-term)
-*pTet-EYFP
-*pBAD-TetR
-*riboswitch in pSB1A3
-<html>
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-/*!!!!!!!!!!! QuickMenu Styles [Please Modify!] !!!!!!!!!!!*/
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-	#qm0
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-		background-color:transparent;
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-</head>
-<body>
-<ul id="qm0" class="qmmc">
-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
-	<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
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-</body>
-</html>
-===Aug 27===
-'''Fan'''
-Miniprep of the O/N culture
-[DNA] unknown
-DNA was eluted from the spin column in 50mL milliQ water
-*20µL of each DNA sample was sent to iGEM (MIT)
-'''Kirsten'''
-Gel extraction of GFP, mmS6-dT,CheZ-dT
-Purified riboswitch, sRBS, c-term-dT, N-term-mRBS. Eluted with 50µL of EB
-Into -20°C freezer.
-µL ligations of GFP+dT, sRBS+mmS6-dT and riboswitch+cheZ.  Left at room temp for 2 hours until transformation.
-'''Alix'''
-Transformations of ligations following iGEM protocol
-===Aug 31===
-'''Roxanne'''
-PCR of CFP, ECFP, EYFP w/ n-term prefix or c-term suffix
-µL Restrictions of:
-*pLacI with SpeI,PstI
-*pStrong with SpeI,PstI
-*Lumazine-dT with XbaI ,PstI
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