RIGHT-CLICK 4× DNA
LIGATION MIXTM
|
1. |
Lambda DNA HindIII
digested before reaction |
2. |
Same after 5 min
of ligation |
3. |
Same after 10 min
of ligation |
10 µl of each sample on 1% agarose gel
Same pattern was observed after
purification and redigestion of ligated DNA. |
Cat.# B 0014 Price: £80.00
Pack size: 50 reactions
Storage: -20 °C (3 months),
+4 °C (3 weeks)
Description: The RightClick
4× DNA Ligation MixTM is designed for fast and efficient ligation of
cohesive and blunt-end DNA fragments at room temperature.
Applications: DNA fragment
cloning , vector recircularization, linker ligation.
Content:
Right-Click DNA
Ligase
50 µl
4x Right-Click Ligation
Buffer
250 µl
DNA Dilution Buffer
(Tris-Cl 10 mM, EDTA 0.1 mM, pH7.8) 500 µl
Right-Click DNA Ligase – T4
DNA Ligase at optimised concentration.
4x Right-Click Ligation Buffer –
Mix containing Mg2+, ATP, Tris-buffer, and additives.
Note: Mix thoroughly before
use. Avoid multiple freeze/thaw cycles.
Quality Controls:
- Lambda DNA samples
(100 ng each) were cut with EcoRV (blunt), and Hind III
(cohesive), and then religated into fragments bigger than 12kb and 25kb
respectively. Time needed for that was less than 20 min and 5 min
respectively.
- Loss of transformation
efficiency comparing with classic 16h at +4°C ligation was no more than 20%.
Shipping: Any temperature
equal or below ambient.
Product use and limitation:
Usage
|
Length of DNA fragments
|
2kb – 6kb |
6kb – 10kb |
>10kb |
sticky |
blunt |
sticky |
blunt |
sticky |
blunt |
Maximum amount to be ligated |
100 ng |
100 ng |
150 ng |
150 ng |
200 ng |
200 ng |
Time needed for DNA ligation
by 90% |
5 min |
20 min |
10 min |
40 min |
20 min |
60 min |
Maximum value for
vector/insert ratio* |
1/5 |
1/10 |
1/10 |
1/20 |
1/20 |
1/50 |
Maximum duration of reaction
before it will reduce consequent transformation efficiency** |
1 h |
1 h |
1 h |
* Data is obtained for ligation of
fragments that were cloned before and are used for recloning procedures. We
recommend increasing the number of inserts by up to 10 times when PCR products are
involved in reaction.
**Efficiency of transformation usually decreases if
the reaction is longer than 1 h.
Dephosphorylation of vector is
advised in case of blunt-end ligation (1u of CIAP per 100-500 ng of DNA, 20 min
at 37 ºC, 20 min at 80 ºC, following purification with DNA purification kit or
with Phenol/Chloroform is optional but recommended).
RIGHT-CLICK 4× DNA LIGATION
PROTOCOL
- Estimate number of
reactions (N): do not forget to include control ligations. Final volume
of one reaction is 20 µl.
- Preparation of
4× Ligation Mix: take N µl of Ligase and 5× N µl of
Ligation Buffer, mix thoroughly by pipetting. We recommend using freshly prepared
4× Mix, it can be also stored at +4ºC for up to 3 days.
- Take 5 µl of 4× Mix in a
1.5 ml sterile Eppendorf tube at room temperature, add 15 µl of DNA solution
containing vector and insert with gentle up/down pipetting up to 20 times.
Do not allow bubbling.
- Leave for suggested
time (see Usage Tips). Allow more time if more DNA is used in reaction than
advised. Do not exceed 1h.
- Optional step:
preheating for 20 min at 65 ºC. It does not affect results of ligation or
following transformation.
- Take 2 µl of reaction
mix for control electrophoresis (highly recommended).
- Add 100 µl of competent
cells, proceed to transformation procedure according to the protocol from your
supplier of competent cells.
- Run agarose
electrophoresis with 2 µl of all control and experimental reactions.
Typical
scheme of ligation procedure:
Experiment reaction |
Control 1 |
Control 2 |
Control 3
|
Control 4 (optional) |
Vector X µl |
Vector X µl |
Vector X µl |
--- |
--- |
Insert Y µl |
--- |
Insert Y µl |
Insert Y µl |
--- |
4×Mix 5 µl |
4× Mix 5 µl |
--- |
4× Mix 5 µl |
--- |
ddH2O up to 20µl |
ddH2O up to 20µl |
ddH2O up to 20µl |
ddH2O up to 20µl |
ddH2O 20µl |
If
vector was dephosphorylated, we advise you to do a control transformation
with same amount of vector before phosphorylation.
TROUBLESHOOTING
1. No
colonies:
-
Check
antibiotic;
-
Reaction is inhibited by DNA impurities – purify DNA;
-
Vector is degraded or damaged (often after over-dephosphorylation) – run
electrophoresis;
-
Vector or DNA are toxic for cells – review strategy for cloning;
-
Competent cells are dead – use another stock or fresh competent cells;
-
Amount of vector is low – add more vector DNA.
- Background (number of
non-recombinant colonies) is high:
- Vector is not fully
digested or needs dephosphorylation – digest vector again and
dephosphorylated, purify;
- Concentration of insert
is low - increase amount of insert DNA in reaction;
- Competent cells are
contaminated – use reliable stock of cells;
-
Antibiotic
level is low – prepare fresh plates or/and increase antibiotic concentration.