Experiment
Transformation ( by 1.5 ml Eppendorf tube)
1. 2.5 μg DNA + 25 μl competent cell (ECOS 101 DH5α)
2. On ice 30 min
3. 42℃ heat shock 30 sec
4. On ice 5 min
(In laminar flow)
5. Add LB to 500 μl，37℃ 170 rpm incubate 2hour.
6. plate 200 μl ( 300 μl remained) spread over plate by spreader.
7. Incubate the plate in 37℃ overnight.
Patch and small scale culture
1. Pick a colony by toothpick then “patch” on another agar plate as bacteria stock. Incubate in 37℃ 14 hour~16 hour.
EX: picture
2. Dip the toothpick into 5 ml LB (with antibiotic) as small scale culture for plasmid extraction. Incubate in 37℃ 170rpm for 14 hour~16 hour.
Mini extraction
Follow the “Biokit” protocol.
IV. Digestion
1. 0.5 μg DNA + 0.2 μl fast digestion restriction enzyme ( 10 unit/μl, final 0.25 μg/unit) + 1 μl 10X buffer + mQ H2O -> total volume 10 μl.
2. Bath in 37℃ warm water for 15 min.
Ligation protocol
*The ideal volume for DNA ligation is 10 μl, but it is adding the volume to 15~20 μl is fine, since part of the liquid will vaporize during ligation.
*It is recommended that the concentration for the backbone is 20 fmol.
*The molar ratio for backbone : insert = 1 : 3
*The ligase required 0.1 ~ 0.5 unit/tube.
1. Quantify the concentration of the linear DNAs that you are going to ligate.
2. Calculate the molar weight of DNA. Converse the unit into ng/fmol.
3. Calculate how much Nano gram is required for 20 fmol of backbone and 60 fmol of insert.
4. Then by the quantified concentration, find out that how much μl is required.
5. Adjust the total volume to 8.9 μl/tube.
6. Add 1 μl of 10x ligation buffer and 0.1 μl of T4 ligase (5 unit/μl)
7. Place it in 16°C water bath, overnight.
Run electrophoresis gel
1. X (g) agarose + Y (ml) 0.5XTAE make a suitable agarose gel( depend on your DNA size), X/Y = 1.2%~1.5% (<0.5k) heating until all agarose melted.
0.8%~1% (0.5~10k)
0.3%~0.7% (>10k)
Cool down the agarose gel (use the back of your hand to check it, as long as it does not burn your hand, it is suitable temperature) + EtBr ( final, 5 μl/100 ml).
2. Marker:
| Marker | Sample | |
|---|---|---|
| Suitable ladder | DNA | |
| 2 μl | 10 μl | |
| 6X dye | 2 μl | 2 μl |
| mQ H2O | 8 μl | 0 |
| Total volume | 12 μl | 12 μl |
3. Load 12 μl per well, then run gel in electrophoresis tank under 100 V.
PCR
Purpose: to check whether our plasmid has successfμlly transformed into E.coli, DH5α.
1. PCR condition finding
* P.S. Prepare the stock solution first, which consist of mQH2O、10x buffer、forward primer、backward primer、dNTP and DNA, then add the DNA polymerase before starting the PCR process.
* 0.5 ~ 1 tube shall be added to the ideal amount when preparing the stock solution.
It is recommended to dilute the amount polymerase needed to 1 unit/l, to decrease the error of adding small amount of volume.
Recipe
| DNA (10 ng) | 10 ng |
|---|---|
| 10x buffer | 5 μl |
| forward primer (2.5 μM) | 2.5 μl |
| Reverse primer (2.5 μM) | 2.5 μl |
| dNTP (10 μM) | 1 μl |
| DNA polymerase (5 unit/μl) | 1~2 unit |
| Total volume , add water to 50 μl | 50 μl |
| Final concentration | |
|---|---|
| DNA | 10 ng/ 50 μl |
| Buffer | 1x |
| Forward primer | 0.125 μM |
| Backward primer | 0.125 μM |
| dNTP | 0.2 μM |
| DNA polymerase | 1~2 unit |
2. Condition entering
a. 94°C for 1 min.
b. 94°C for 30 sec
c. The specific temperature for primer to bind on DNA, 30 sec (pSBBS1C 55°C, pSBBS4S 65°C)
d. 72°C for 1 min
e. GOTO 2, 25 cycle
f. 16°C for 10 min (to cool the machine)
g. Run gel
h. Result:
a. pSBBS1C
