Team:PKU Beijing/Notebook/AND Gate 1/Core/Sal T7p

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Notebook > AND Gate 1 > Core > Molecular cloning for AND GATE: Sal_T7ptag + AraC_SupD

Molecular cloning for AND GATE: Sal_T7ptag + AraC_SupD

2009.8.26

Receive Sal insert digested by two enzymes EcoRI and SpeI, several low-copy backbone plasmids (1-9C,Tc+;1-7G,K+;1-1M,A+) and AraC_SupD plasmid.

Digest 1-9C backbone plasmid into vector

1.5μLEcoRI
1.5μLXbaI
2μL10×M buffer
7μLplasmid
8μLddH2O
20μLTotal

2009.8.27

Electrophoresis to identify 1-9C vector digested from the plasmid
Result (from left to right)
1-9C①, 1-9C plasmid control①, Marker, 1-9C②, 1-9C plasmid control②
PKU 20090827 Guosheng Zhang 1.JPG
The result is strange, change another backbone

Digest 1-7G backbone plasmid into vector

1.5μLEcoRI
1.5μLXbaI
2μL10×M buffer
5μLplasmid
10μLddH2O
20μLTotal

Electrophoresis to identify 1-7G vector digested from the plasmid
Result (from left to right)
Marker, 1-7G①, 1-7G②, 1-7G plasmid control
PKU 20090827 Guosheng Zhang 2.JPG
The concentration of 7G plasmid is too low, so the electrophoretic bands are invisible.

2009.8.28

Digest 1-7G backbone vector again and electrophoresis to identify
Result (from left to right)
1-7G, 1-7G plasmid control, Marker,
PKU 20090828 Guosheng Zhang 1.JPG
Still failed.

Digest 1-1M backbone plasmid into vector

1.5μLEcoRI
1.5μLXbaI
2μL10×M buffer
15μLplasmid
0ddH2O
20μLTotal

Electrophoresis to identify 1-1M vector digested from the plasmid
Result (from left to right)
1-1M, 1-1M plasmid control, Marker
PKU 20090828 Guosheng Zhang 2.JPG

DNA purification to extract 1-1M vector

Ligate the Sal insert and 1-1M vector
System

3μLSal insert
1μL1-1M vector
1μL10× Ligase buffer
1μLLigase
4μLddH2O
10μLTotal

Transformation for the ligation product: Sal insert + 1-1M vector

2009.8.29

A mistake was made when digesting 1-1M vector: there is a RFP part between restriction site XbaI and SpeI, so 1-1M vector should be digested by EcoRI and SpeI!

Digest 1-1M vector again

1.5μLEcoRI
1.5μLSpeI
2μL10×H buffer
15μLplasmid
20μLTotal

DNA purification to extract 1-1M vector, ligate the Sal insert and 1-1M vector and transform the ligation product: Sal insert + 1-1M vector

2009.8.31

Miniprep for Sal+1M plasmid

Digest Sal+1M vector

1.5μLSpeI
1.5μLPstI
2μL10×H buffer
6μLplasmid
9μLddH2O
20μLTotal

The electrophoretic result is strange, so prepare to miniprep again tomorrow.

2009.9.1

Miniprep for Sal+1M plasmid again

Determine the concentration of T7ptag plasmid by spectrophotometer
Sal+1M plasmid A: 5.0174ng/μL×50=250.87ng/μL
Sal+1M plasmid B: 4.1509ng/μL×50=207.55ng/μL
Sal+1M plasmid C: 3.2412ng/μL×50=162.06ng/μL

Digest Sal+1M vector again

Electrophoresis to identify the Sal+1M vector
Result (from left to right)
Sal_1M A, Sal_1M B, Sal_1M C, plasmid control, Marker
PKU 20090901 Guosheng Zhang 1.JPG
The positions of three digestion products are a little different, so digest the plasmid with XbaI and PstI to identify.

1μLSpeI
1μLPstI
1μL10× buffer
3μLplasmid
4μLddH2O
10μLTotal

Electrophoretic result (from left to right)
Sal_1M A, Sal_1M B, Marker, Sal_1M C
PKU 20090901 Guosheng Zhang 2.JPG
Ligate the RBS+T7ptag+Terminator insert and Sal+1M vector
System

3μLinsert
1μLvector
1μL10× Ligase buffer
1μLLigase
4μLddH2O
10μLTotal

2009.9.2

Transformation for the ligation product: RBS+T7ptag+Terminator insert and Sal+1M vector

Identify the colonies on the plates (Sal_1M+T7ptag) by PCR
System

5μLMaster mix
0.25μLForward primer
0.25μLReverse primer
4.5μLddH2O
Template (Bacteria Colony)
10μLTotal

2009.9.3

Electrophoresis to identify the PCR product
Result 1 (from left to right, from top to bottom)
1H①~⑤,1J①~④,Marker,1J⑤,2G①~⑤,2I①~②
2I③~⑤, 2K①~⑤,Marker, 2M①,Marker,2M②~⑤,5J①~④
PKU 20090903 Guosheng Zhang 1.JPG
Result 2 (from left to right)
5J⑤,5N①~⑤,Marker,11N①~⑤
PKU 20090903 Guosheng Zhang 2.JPG
Choose 27 samples to miniprep
1H②④⑤,1J①②④,2G②③④,2I②③⑤,2K①②③,
2M①②③,5J③④⑤,5N①②④,11N①②⑤

Double digestion for Sal+T7ptag insert

1μLXbaI
1μLPstI
2μL10×M buffer
5μLSal+T7ptag plasmid
11μLddH2O
20μLTotal

Double digestion for AraC_SupD vector

1.5μLSpeI
1.5μLPstI
2μL10×H buffer
4μLplasmid
11μLddH2O
20μLTotal

Electrophoresis and Gel Extraction for Sal+T7ptag insert
Result (from left to right, from top to bottom)
1H②④⑤,1J①②④,2G②,Marker,2G③④,2M①②③,2I②③
2I⑤,2K①②③,5J③④⑤,Marker,5N①②④,11N①②⑤,AraC_SupD vector
PKU 20090903 Guosheng Zhang 3.JPG
Extract 1H⑤,2G②,2I③,2K②,5J④,5N① from the gel
DNA purification to extract AraC_SupD vector

Ligate the Sal_RBS_T7ptag insert and AraC_SupD vector
System

3μLinsert
1μLvector
1μL10× Ligase buffer
1μLLigase
4μLddH2O
10μLTotal

2009.9.4

Transformation for the ligation product: Sal_RBS_T7ptag insert + AraC_SupD vector

Choose three other colonies for 5N, 11N, 1J, 2M and shake them
12 samples in total: 5NA\B\C, 11NA\B\C, 1JA\B\C, 2MA\B\C
Miniprep for 12 samples

2009.9.5

Double digestion for Sal_RBS (5N, 11N, 1J, 2M)_T7ptag insert

1.5μLXbaI
1.5μLPstI
2μL10×M buffer
5μLplasmid
10μLddH2O
20μLTotal

Electrophoretic result (from left to right)
5NA\B, 1JB\C, Marker, 11NA\B, 2MB\C
PKU 20090905 Guosheng Zhang 1.JPG
None correct ligation products can be extracted.

Double digestion for Sal_RBS (1H, 2G, 2I, 2K, 5J, 5N)_T7ptag+AraC_SupD insert

1.5μLEcoRI
1.5μLPstI
2μL10×H buffer
4μLplasmid
11μLddH2O
20μLTotal

Electrophoresis and Gel Extraction for Sal_RBS (1H, 2G, 2I, 2K, 5J, 5N)_T7ptag+AraC_SupD insert
Result (from left to right)
1H①②③, 2I①②③, 2G①, Marker, 2G②③, 5J①②③, 2K①②③
PKU 20090905 Guosheng Zhang 2.JPG
Extract 1H②,2I②,2G①,5J②,2K① from the gel

2009.9.6

Ligate the Sal_T7ptag+AraC_SupD insert(EP) and 1-7G(K+) backbone
System

5μLinsert
1μLbackbone
1μL10× Ligase buffer
1μLLigase
2μLddH2O
10μLTotal

I mistake the plasmid for backbone, so ligate again and transform the ligation product.

I made a great mistake, the length of Sal_RBS_T7ptag+AraC_SupD insert should be a little larger than 5k and the backbone be 3k, which is not in accordance with the results I got yesterday, indicating that the ligation products are abnormal, so I have to rework.

Double digestion for Sal_RBS (1H⑤, 2G②, 2I③, 2K②, 5J④)_T7ptag insert again

1.5μLXbaI
1.5μLPstI
2μL3 buffer
5μLplasmid
10μLddH2O
20μLTotal

Shift AraC_SupD to low-copy backbone 1-7G

1.5μLEcoRI
1.5μLPstI
2μLEcoRI buffer
4μLplasmid
11μLddH2O
20μLTotal

Electrophoresis and Gel Extraction for Sal_RBS_T7ptag insert
Result (from left to right)
1H, 2G, 2I, 2K, 5J, Marker, AraC_SupD
PKU 20090906 Guosheng Zhang 1.JPG

2009.9.8

Miniprep for 1J①②③, 5N①②③, 11N①②③, 2M①②③(Sal_RBS_T7ptag)

Ligate the AraC_SupD insert(EP) and 1-7G(K+) backbone again.

2009.9.9

Double digestion for Sal_RBS (1J, 5N, 11N, 2M)_T7ptag insert

1.5μLXbaI
1.5μLPstI
2μL3 buffer
6μLplasmid
9μLddH2O
20μLTotal

Electrophoresis and Gel Extraction for Sal_RBS_T7ptag insert
Result (from left to right)
1J①②③, 2M①②③, Marker, 5N①②③, 11N①②③
PKU 20090909 Guosheng Zhang 1.JPG

2009.9.10

Electrophoresis and Gel Extraction for Sal_RBS_T7ptag(2G/5J) insert
Result (from left to right)
2G, Marker, 5J
PKU 20090910 Guosheng Zhang 1.JPG
There is something wrong with 2G insert, so only extract 5J inset.
Gel: Sal_5J_T7ptag insert 0.025gPN volume: 75μL

Receive reverse mutated T7ptag bacteria liquid from Lin Min, shake it and store the strain.
Miniprep for T7ptag plasmid
Determine the concentration of T7ptag plasmid by spectrophotometer
4.6422ng/μL×50=232.11ng/μL



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