Cloning strategies

1
Cloning strategies

• Construction
• Completely define compatible vector and insert
  (s)
• Design
• Define vector
• Define feature (s) to be inserted
• Let Vector NTI design the best cloning strategy

2
Construction

• Vector pUC19 (Ecor1-Sma1)
• Insert pBR322 (Ecor1-Ava1)

3
Define the vector (recipient fragment)
Shift click
4
Define the insert (donor fragment)
Shift click
5
Generate the construct
6
Modify the insert
7
Original vectors and new construct
8
Design1

• Vector pUC19
• 5 nucl.500
• 3 nucl. 250
• Feature to be inserted TC(R) of pBR322
• 5 undefined
• 3 undefined

9
Define the vector (recipient fragment)
10
Define the insert (donor fragment)
11
Set up design parameters
12
Designed molecule
13
Design2

• Vector BPV1
• Open at ApaL1, site saved on 5
• Feature to be inserted SV40 Large T
• 5 and 3 undefined

14
Define the vector (recipient fragment)
15
Define the insert (donor fragment)
16
Designed molecule
17
Design3

• Vector BPV1
• 5 nucl. 5000
• 3 nucl. 2500
• Feature to be inserted SV40 largeT
• 5 undefined (444 flanking sequence)
• 3 NCO1
• inverted

18
Define the vector (recipient fragment)
19
Define the insert (donor fragment)
20
Modify insert
21
Designed molecule
22
Use of linker/adaptor
23
Other functionalities

• Topo cloning (user manual 337)
• Gateway cloning (user manual 321)

24
Gel display
25
Gel setup
26
Gel sample
27
Gel marker
28
Label a fragment
Right click
29
Separation of fragments
30
Use of fragment list
Load from list
31
ContigExpress
32
Open/create a project
33
Assemble sequences
34
Assembly setup
35
Project display options
36
Find a fragment
37
Fragment display
Double click
38
Fragment display options
39
Select a sequence
40
Assembly display
Assemble selected/non-selected fragments
41
Contig view options
Contig coverage
42
Find fragment
43
Display options
44
Ambiguities display
45
Edit a fragment
46
Sequence comparison
Right click
47
Reference sequence
Right click
48
End trimming
49
Vector trimming
50
Vector trimming settings
51
Secondary peaks
52
Exercise 6

• Open DemoProject.cep
• Trim ends of all sequences
• Edit pane layout (standard)
• Set ONE15F as reference sequence in contig 1
  (select Edit Mode)
• Display amino acid residues onto consensus
• Compare ONE13R and consensus.