Designing loop primers
1. Enter the target sequence information
Upload the primer information file for the loop primer designing, which was saved in the regular primer designing.
Push [Browse] button to select the Primer information file, which was saved from the page of Detail information of the loop primer sets.
Users cannot select their Parameter set.
Select the Primer Information file and push the [Primer Design] button to proceed to the Basic designing page.
2. Loop primer design
Basic procedure for the loop primer design is as follows.
- Click [Generate] button to generate the loop primer candidates.
- Click [Display] button to display the loop primer list.
2-1. Basic designing page
The explanations of items on the page are listed below.
Target sequence display
- Sequences are usually displayed in capital letters.
Message area
The status of the designing process is shown.
| (1)[Generate] | Generate the primer sets. |
|---|---|
| (2)[Display] | List the generated primer sets in a new window. |
| (3)Show Page | Select the page number for the display of primer list in a new window. |
| (4)[Detail Settings] | Shift to Detail Setting page. |
2-2. Detail Settings page
The explanations of items on the page are listed below.
Target sequence display
- Sequences are usually displayed in capital letters.
Message area
The status of the designing process is shown.
| (1)[Generate] | Generate the primer sets. |
|---|---|
| (2)[Display] | List the generated primer sets in a new window. |
| (3)Show Page | Select the page number for the display of primer list in a new window. |
| (4)[Basic Designing] | Shift to Basic Designing page. |
| (5)[Reset Parameter] |
Reset parameter value to the default condition. |
| Specify the parameter setting by inputting the appropriate number or clicking on [-] or [+] button beside the number entry field to change the input value. | |
| (6)Length | Minimum and maximum length of the loop primer. |
| (7)Tm | Minimum and maximum Tm value of the loop primer. |
| (8)GC rate(%) | Acceptable value for GC rate. |
| (9)dG threshold[Kcal/mol] |
3'stability Dimer check |
| (10)Limitations |
Maximum number of combinations when primer set is generated. LF/LB Sets |
| [Regular primer] | |
| (11)Regular Primer dG threshold[Kcal/mol] |
dG threshold 5' stability 3'stability |
3. Loop primer list
Up to 100 generated loop primer sets will be listed in each page.
Each row stands for one loop primer set, and each loop primer fragment's location refers to the target sequence above directly.
After pushing [Confirm] button, the selected loop primer sets will be shown in a new window for Detail information of the loop primer sets.
When push [Save List] button, the loop primers listed on the screen will be saved as an Excel file.
4. Detail information of the loop primer sets
The loop primer's information below is shown.
- LF
- LB
The following information is shown with each loop primer fragment.
- Name
- The position of 5' end on the target sequence
- The position of 3' end on the target sequence
- Length
- Tm value
- Stability of 5' end
- Stability of 3' end
- GC rate
- Loop Primer sequence
PrimerExplorer Operation Instructions
Introduction
Operation of each screen
Appendix
