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-	'''Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD'''	+	{| border="0" cellspacing="8px" cellpadding="15" width="80%"
		+	|-
		+	|[[Berkeley_LBL|Home]]
		+	|[[Berkeley_LBL/Project|Project Description]]
		+	|[[Berkeley_LBL/Methods|Methods]]
		+	|[[Berkeley_LBL/Notebook|Notebook]]
		+	|[[Berkeley_LBL/Results|Results and Discussion]]
		+	|[[Berkeley_LBL/Resources|Resources]]
		+	|}
-	'''''Cyanobacteria - Synechocystis'''''	+	== '''Construction of pET3A Derivatives Containing R-bchH, R-bchI, and R-bchD''' (Simultaneous Ligation) ==
-	'''S-chlH:''' 3996 base pairs	+
-	'''S-chlI''': 1074 base pairs	+
-	'''S-chlD''': 2031 base pairs	+
-	[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides]]
-	'''pET3A:'''	+	'''''Rhodobacter sphaeroides'''''
		+	'''R-bchH:''' 3582 base pairs
		+	'''R-bchI''': 1005 base pairs
		+	'''R-bchD''': 1695 base pairs
-	1. Innoculate ''pET3A'' single colony from plate into 250 mL flask containing 50 mL LB + 50 ul Carb.  Allow to grow in 30°C shaker overnight.	+	[[Berkeley_LBL/MimiSequence2|Sequences and Properties of Oligonucleotides for R-bch Genes]]
-	2. [[Berkeley_LBL/Miniprep|Miniprep]] cultures.	+	[[Berkeley_LBL/Mimi_RbchHID|'''Construction of ''pET3A-(R)-bchHID''''']]
		+	- Not Successful
-	3. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A'' with enzymes''NdeI'' and ''BamHI''using the following conditions:	+	[[Berkeley_LBL/MimiSequence3|Sequences and Properties of Oligonucleotides for R-bch Genes(NEW)]]
-	42.1 ul pET3A plasmid	+	[[Berkeley_LBL/Mimi_RbchHID2|'''Construction of ''pET3A-(R)-bchHID''' - 2nd Attempt'']]
-	5 ul NEB 4 (10x)	+	- Not Successful
-	0.5 ul BSA (100x)	+
-	1.2 ul NdeI	+
-	1.2 ul BamHI	+
-		+
-	'''Construction of ''pET3A-(S)-chlH'':'''
-	1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
		+	== '''Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD''' (Sequential Ligation) ==
-	Amplification introduces sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
-	2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~4kb).	+	'''''Cyanobacteria Synechocystis'''''
		+	'''S-chlH:''' 3996 base pairs
		+	'''S-chlI''': 1074 base pairs
		+	'''S-chlD''': 2031 base pairs
-	3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]	+	[[Berkeley_LBL/MimiSequence|Sequences and Properties of Oligonucleotides for S-chl Genes]]
-	4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:	+	[[Berkeley_LBL/Mimi-SchlH|'''Construction of ''pET3A-(S)-chlH''''']]
-		+	[[Berkeley_LBL/Mimi-SchlI|'''Construction of ''pET3A-(S)-chlHI''''']]
-	Digest in 37°C for 2 hours.	+
-	Add 0.5 ul NdeI and 0.5 ul BamHI.	+	[[Berkeley_LBL/Mimi-SchlD|'''Construction of ''pET3A-(S)-chlHID''''']]
-	Digest in 37°C for additional 30 minutes.	+
-		+
-	5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.	+
-		+
-	6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band.	+
-		+
-	7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"	+
-		+
-	8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:	+
-		+
-		+
-	Plate onto LB Agar + Carb plate	+
-		+
-	9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.	+
-		+
-	10. [[Berkeley_LBL/Miniprep|Miniprep]] cultures	+
-		+
-	11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:	+
-		+
-	20 ul DNA	+
-	3 ul NEB 4 (10x)	+
-	1 ul NdeI	+
-	1 ul SpeI	+
-	3 ul BSA (10x)	+
-	2.0 ul H2O	+
-	-------------	+
-	30 ul total	+
-		+
-	Run gel – look for ~4kb and ~5kb band	+
-		+
-	Save glycerol stocks	+
-		+
-		+
-	'''Construction of ''pET3A-(S)-chlHI'':'''	+
-		+
-	1. Amplify Synechocystis-Cyanobacteria gene ''S-chlI'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:	+
-		+
-		+
-	Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene.	+
-		+
-	2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~1kb).	+
-		+
-	3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]	+
-		+
-	4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlI'' with ''KpnI'' and ''BglII'' using the following conditions:	+
-		+
-		+
-	Digest in 37°C for 2 hours.	+
-	Add 0.5 ul KpnI and 0.5 ul BglII.	+
-	Digest in 37°C for additional 30 minutes.	+
-		+
-	5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.	+
-		+
-	6. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlH'' with ''KpnI'' and ''BamHI'' using the following conditions:	+
-		+
-		+
-	Digest in 37°C for 2 hours.	+
-	Add 0.5 ul KpnI and 0.5 ul BglII.	+
-	Digest in 37°C for additional 30 minutes.	+
-		+
-	6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions.	+
-		+
-	7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''"	+
-		+
-	8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:	+
-		+
-		+
-	Plate onto LB Agar + Carb plate	+
-		+
-	9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.	+
-		+
-	10. [[Berkeley_LBL/Miniprep|Miniprep]] cultures	+
-		+
-	11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:	+
-		+
-	20 ul DNA	+
-	3 ul NEB 1 (10x)	+
-	1.4 ul SpeI	+
-	0.9 ul KpnI	+
-	3 ul BSA (10x)	+
-	1.7 ul H2O	+
-	-------------	+
-	30 ul total	+
-		+
-	Run gel – look for ~1kb and ~9kb band	+
-		+
-	Save glycerol stocks	+
-		+
-		+
-		+
-	'''Construction of ''pET3A-(S)-chlHID'':'''	+
-		+
-	1. Amplify Synechocystis-Cyanobacteria gene ''S-chlD'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:	+
-		+
-	''PCR:''	+
-	1 ul Schl-D	+
-	10 ul HF Buffer 5x	+
-	1 ul dNTP	+
-	5 ul primer mix	+
-	0.5 ul Phusion	+
-	32.5 ul H2O	+
-	--------------	+
-	50 ul total	+
-		+
-	Conditions:	+
-	98°C 30s	+
-	98°C 8s	+
-	61°C 30s	+
-	72°C 1:10m	+
-	Go to 2 for additional 29 cycles	+
-	72°C 10m	+
-	4°C         ---	+
-		+
-		+
-	Amplification introduces sites ''SpeI-rbs'' and ''NotI-BglII'' into the gene.	+
-		+
-	2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~2kb).	+
-		+
-	3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]	+
-		+
-	4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlD'' with ''SpeI'' and ''NotI'' using the following conditions:	+
-		+
-	''Schl-D Sequential Restriction Digestion:''	+
-		+
-	''Digestion #1''	+
-	43 ul Schl-D	+
-	5 ul NEB 2 (10x)	+
-	0.5 ul BSA (100x)	+
-	1.5 ul SpeI	+
-		+
-	2 hour digestion in 37°C	+
-	Add 0.5 ul SpeI	+
-	30 min digestion in 37°C	+
-	[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]	+
-		+
-	''Digestion #2''	+
-	43 ul Schl-D	+
-	5 ul NEB 3 (10x)	+
-	0.5 ul BSA (100x)	+
-	1.5 ul NotI	+
-		+
-	2 hour digestion in 37°C	+
-	Add 0.5 ul NotI	+
-	30 min digestion in 37°C	+
-		+
-	5. Added a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker	+
-	[[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion.	+
-		+
-	6. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlHI'' with ''SpeI'' and ''NotI'' using the following conditions:	+
-		+
-	''Sequential Restriction Digestion for pEt3A-(S)-HI:''	+
-		+
-	''Digestion #1:''	+
-	43 ul pEt3A-(S)-HI	+
-	5 ul NEB 2 (10x)	+
-	0.5 ul BSA (100x)	+
-	1.5 ul SpeI	+
-		+
-	2 hour digestion in 37°C	+
-	Add 0.5 ul SpeI	+
-	30 min digestion in 37°C	+
-	[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]	+
-		+
-	''Digestion #2:''	+
-	43 ul pEt3A-(S)-HI	+
-	5 ul NEB 3 (10x)	+
-	0.5 ul BSA (100x)	+
-	1.5 ul NotI	+
-		+
-	2 hour digestion in 37°C	+
-	Add 0.5 ul NotI	+
-	30 min digestion in 37°C	+
-		+
-	6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~2kb and ~10kb).	+
-		+
-	7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlD'' to plasmid ''pET3A-(S)-chlHI", yielding plasmid "'''pET3A-(S)-chlHID'''" using the following conditions:	+
-		+
-	12 ul pET3A-(S)-HI	+
-	4 ul Schl-D	+
-	2 ul Ligase Buffer	+
-	1 ul Ligase Enzyme	+
-	1 ul H2O	+
-	-------------------	+
-	20 ul total	+
-		+
-	8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:	+
-		+
-	7 ul pET3A-(S)-HID ligation	+
-	73 ul H2O	+
-	20 ul KCM solution	+
-	100 ul Chemical Competent Novablue cells	+
-	-----------------------------------------	+
-	200 ul total	+
-		+
-	Plate onto LB Agar + Carb plate	+
-		+
-	9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.	+
-		+
-	10. [[Berkeley_LBL/Miniprep|Miniprep]] cultures	+
-		+
-	11. [[Berkeley_LBL/Digestion2|Analytic Digestion]] using the following conditions:	+
-		+
-	20 ul DNA	+
-	3 ul NEB 2 (10x)	+
-	1.8 ul NotI	+
-	1 ul SpeI	+
-	3 ul BSA (10x)	+
-	1.2 ul H2O	+
-	-------------	+
-	30 ul total	+
-		+
-	Run gel – look for ~2kb and ~9kb band	+
-		+
-	Save glycerol stocks	+

---

Latest revision as of 20:29, 26 October 2007

Home

Project Description

Methods

Notebook

Results and Discussion

Resources

Construction of pET3A Derivatives Containing R-bchH, R-bchI, and R-bchD (Simultaneous Ligation)

Rhodobacter sphaeroides

       R-bchH: 3582 base pairs
       R-bchI: 1005 base pairs
       R-bchD: 1695 base pairs

Sequences and Properties of Oligonucleotides for R-bch Genes

Construction of pET3A-(R)-bchHID - Not Successful

Sequences and Properties of Oligonucleotides for R-bch Genes(NEW)

Construction of pET3A-(R)-bchHID - 2nd Attempt - Not Successful

Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD (Sequential Ligation)

Cyanobacteria Synechocystis

       S-chlH: 3996 base pairs
       S-chlI: 1074 base pairs
       S-chlD: 2031 base pairs

Sequences and Properties of Oligonucleotides for S-chl Genes

Construction of pET3A-(S)-chlH

Construction of pET3A-(S)-chlHI

Construction of pET3A-(S)-chlHID