Notebook entry
Notebook entry
Tuesday, 12/15/2015
(Deleted)
The DNA sequence corresponding to this note no longer exists.
Figure 1. DsRed2 cloning into pDusk vector (12/15/2015)
Materials
Table1
A A | B B | C C | D D | E E | |
1 1 | Number of Reactions | 80 | 40 | 20 | |
2 2 | Component | Amount (uL/tube) | Master Mix (uL) | Master Mix (uL) | Master Mix (uL) |
3 3 | Water | 16 | 1280 | 640 | 320 |
4 4 | 10x Buffer | 2 | 160 | 80 | 40 |
5 5 | Enzyme | 1 | 80 | 40 | 20 |
6 6 | Master Mix Total | 19 | 1520 | 760 | 380 |
7 7 | Template DNA | 1 | |||
8 8 | Reaction Total | 20 |
Wednesday, 12/16/2015
Combine above reaction in tube
Incubate at 37°C for an hour
Run gel
Extract band and save
Reference plasmid map pDusk-DsRed2 tomorrow.
Thursday, 12/17/2015
Drag and drop an image in to view it inline and take notes about it:
pAS329_ligation_check.png
pAS329_ligation_check.png
Lane 1: DNA ladder
Lane 2: undigested
Lane 3: digested vector
Lane 4: insert DNA
Lane 5: ligation tube
Friday, 12/18/2015
Link to other Benchling files by dragging them in from the file browser at the left.
pBR322
pBR322
pBR322 is the backbone I'm going to use for this experiment.
The ampR gene is penicillin beta-lactamase.
It also has the tetR gene and the rop gene.
Saturday, 12/19/2015
1.
Add nuclease
Benchling_Marketing_1-pager.pptx
Benchling_Marketing_1-pager.pptx
Friday, 8/12/2016
Protocol: pDusk-DsRed2 Cloning
Introduction
This protocol is for cloning pDusk +DsRed2.
Materials
Procedure
- Digest and dephosphorylate 5 μg of pDusk plasmid with BbsI for 30 min at 37°C.
Table1
A A | B B | |
1 1 | Ingredient | Amount |
2 2 | Plasmid concentration (μg/μL) | 1 |
3 3 | Plasmid (μg) | 1 |
4 4 | Plasmid volume (μL) | 1 |
5 5 | FastDigest BbsI | 1 |
6 6 | FastAP | 1 |
7 7 | 10X FastDigest Buffer | 2 |
8 8 | ddH2O | 15 |
9 9 | Total (μL) | 20 |
- Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB. (See protocol.)
- Phosphorylate and anneal each pair of oligos:
Table2
A A | B B | |
1 1 | Number of pairs | 5 |
Table3
A A | B B | C C | |
1 1 | Amount per rxn (μL) | Master Mix (μL) | |
2 2 | 10X T4 Ligation Buffer | 1 | 5 |
3 3 | ddH2O | 6.5 | 32.5 |
4 4 | T4 PNK | 0.5 | 2.5 |
5 5 | Master Mix Total | 8 | 40 |
6 6 | Oligo 1 (100 μM) | 1 | |
7 7 | Oligo 2 (100 μM) | 1 | |
8 8 | Reaction Total | 10 |
- Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).
- Put the phosphorylation/annealing reaction in a thermocycler using the following parameters:
Table4
A A | B B | |
1 1 | 37°C | 30 min |
2 2 | 95°C | 5 min and then ramp down to 25°C at 5°C/min |
- Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.
- Set up ligation reaction and incubate at room temperature for 10 min:
Table5
A A | B B | C C | |
1 1 | Amount | Master Mix (μL) | |
2 2 | BbsI digested plasmid concentration (ng/μL) | 50 | |
3 3 | BbsI digested plasmid (from step 2, ng) | 50 | |
4 4 | BbsI digested plasmid (volume) | 1 | 5 |
5 5 | 2X Quick Ligase Buffer | 5 | 25 |
6 6 | ddH2O | 3 | 15 |
7 7 | Master Mix Total (μL) | 9 | 0 |
8 8 | Oligo duplex (1:200 dilution) (from step 3) | 1 | |
9 9 | Quick Ligase | 1 | |
10 10 | Reaction Total (μL) | 11 |
- (optional) Treat ligation reaction with PlasmidSafe exonuclease to prevent unwanted recombination products.
Table6
A A | B B | C C | |
1 1 | Amount (μL) | Master Mix (μL) | |
2 2 | 10X PlasmidSafe Buffer | 1.5 | 7.5 |
3 3 | 10mM ATP | 1.5 | 7.5 |
4 4 | ddH2O | 1 | 5 |
5 5 | Master Mix Total | 4 | 20 |
6 6 | Ligation reaction (step 4) | 11 | |
7 7 | Reaction Total | 15 |
- Transformation