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

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

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

The result is strange, change another backbone

Digest 1-7G backbone plasmid into vector

Electrophoresis to identify 1-7G vector digested from the plasmid
Result (from left to right)
Marker, 1-7G①, 1-7G②, 1-7G plasmid control

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,

Still failed.

Digest 1-1M backbone plasmid into vector

Electrophoresis to identify 1-1M vector digested from the plasmid
Result (from left to right)
1-1M, 1-1M plasmid control, Marker

DNA purification to extract 1-1M vector

Ligate the Sal insert and 1-1M vector
System

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

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

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

The positions of three digestion products are a little different, so digest the plasmid with XbaI and PstI to identify.

Electrophoretic result (from left to right)
Sal_1M A, Sal_1M B, Marker, Sal_1M C

Ligate the RBS+T7ptag+Terminator insert and Sal+1M vector
System

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

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

Result 2 (from left to right)
5J⑤,5N①~⑤,Marker,11N①~⑤

Choose 27 samples to miniprep
1H②④⑤,1J①②④,2G②③④,2I②③⑤,2K①②③,
2M①②③,5J③④⑤,5N①②④,11N①②⑤

Double digestion for Sal+T7ptag insert

Double digestion for AraC_SupD vector

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

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

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

Electrophoretic result (from left to right)
5NA\B, 1JB\C, Marker, 11NA\B, 2MB\C

None correct ligation products can be extracted.

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

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

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

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

Shift AraC_SupD to low-copy backbone 1-7G

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

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

Electrophoresis and Gel Extraction for Sal_RBS_T7ptag insert
Result (from left to right)
1J①②③, 2M①②③, Marker, 5N①②③, 11N①②③

2009.9.10

Electrophoresis and Gel Extraction for Sal_RBS_T7ptag(2G/5J) insert
Result (from left to right)
2G, Marker, 5J

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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