 |
|
 |
|
|
| Home >
Products >
DNA Modifying Enzymes and Cloning >
Ligases >
Quick Ligation™ Kit > FAQ |  | Quick Ligation™ Kit FAQ
See the DNA Modifying Enzymes and Cloning FAQ also.
Q1: What factors can cause incomplete ligation and/or transformation using the Quick Ligation Kit?
Q2: Why is my transformation not working and what control reactions should be run?
Q3: What is the concentration of T4 DNA Ligase provided in the Quick Ligation Kit?
Q4: Can conventional T4 DNA Ligase (400,000 units/ml) be used with the Quick Ligation buffer?
Q5: How to calculate the molarity of ends?
Q1: What factors can cause incomplete ligation and/or transformation using the Quick Ligation Kit?
A1: The most likely causes are:
a. The Quick Ligation reaction was heat inactivated. Heat inactivation in the presence of PEG, which is contained in the Quick Ligation buffer, inhibits transformation.
b. The Quick Ligation mix was not purified prior to electroporation. PEG in the Quick Ligation buffer prevents electroporation. Ligation products can be purified using a commercially available spin column.
c. The Quick Ligation was incubated overnight. PEG in the Quick Ligation buffer may cause a decrease in transformation efficiency over time. There is no addition benefit of letting the Quick Ligation reaction go beyond 30 minutes.
d. Ligation or transformation was inhibited by high salt in the reaction. Clean up the DNA.
e. Phosphatase (CIP , BAP or SAP) was not completely inactivated or removed following dephosphorylation step. Follow the recommended procedure to remove the phosphatase or use Antarctic Phosphatase (NEB #M0289) which can be completely heat inactivated in 5 minutes at 65°C.
Other factors include:
i. Degraded ATP in buffer older than one year. Add new ATP to a concentration of 1 mM.
ii. Ligation produced only linear molecules because the DNA concentration was too high. We recommend a total DNA concentration of 1-10 µg/ml.
iii. The insert and the vector do not have phosphates.
iv. Too much ligation mixture was added to the cells. We recommend adding 1-5 µl to 50 µl of competent cells.
v. The insert was larger than 10 kb and the conditions didn?t allow circularization. Reduce the insert concentration.
vi. The restriction enzyme did not cleave efficiently. When cleaving near the end of a PCR fragment, we recommend leaving at least 6 base pairs on either side of the restriction site.
vii. The restriction enzyme was not completely inactivated. Phenol/ETOH purify the DNA if the enzyme can not be heat inactivated.
viii. Star activity from the restriction digest cleaved the vector or insert. Check the DNA on a gel. If there are extra bands, reduce the amount of enzyme or time for the restriction digest.
Q2: Why is my transformation not working and what control reactions should be run?
A2: a. The cells are not viable or competent. Run the controls listed below and obtain new cells if needed.
b. The recombinant protein is not well tolerated by E. coli. Try making a fusion with maltose binding protein using the pMAL System (NEB #E8000S). Try another expression system that doesn't involve E. coli.
c. The ligated DNA included inverted and tandem repeats selected against by E. coli. Remove the repeat sequence if possible. Try another expression system that doesn?t involve E. coli.
Note: Use the same DNA concentration for each control, typically 0.1-1 ng per transformation.
i. Uncut vector to check cell viability and test the antibiotic resistance of the plasmid. Plate on media and media plus antibiotic.
ii.Vector cut and not ligated: to check for uncut vector, gel purification may be required to remove the last 0.1% of uncut vector.
iii. Cut and religated plasmid (no insert) to check ligase activity and DNA end integrity. Should be 60-70% of the value obtained for uncut vector.
iv. Cut, dephosphorylated and religated plasmid to check background due to incomplete phosphatase treatment. Should be < 3% of the value obtained for uncut vector.
Q3: What is the concentration of T4 DNA Ligase provided in the Quick Ligation Kit?
A3: 2,000,000 units/ml. It is the same as NEB high concentration T4 DNA Ligase (NEB #M0202).
Q4: Can conventional T4 DNA Ligase (400,000 units/ml) be used with the Quick Ligation buffer?
A4: When using conventional T4 DNA Ligase, increase the reaction time to 15 minutes. Incubations longer than 30 minutes are not recommended.
Q5: How to calculate the molarity of ends?
A5: Molarity = [(µg/µl) ÷ (base pairs x 650 daltons)] x 2 ends
Example: - Calculate the molarity of ends for a linearized 5 kb vector that has a concentration of 250 ng/µl: [(0.25 µg/µl) ÷ (5000 x 650 daltons)] x 2 = 154 nM
- Calculate the molarity of ends if you put 50 ng of this vector in a 20 µl ligation reaction:
50 ng ÷ 20 µl = 0.0025 µg/µl
[(0.0025 µg/µl) ÷ (5000 x 650)] x 2 = 1.54 nM - Determine the amount of a 1 kb insert needed to achieve a 3:1 insert:vector ratio:
3 X 1.54 nM = 4.62 nM
[(X µg/µl) ÷ (1000 x 650)] x 2 = 4.62 nM
0.0015 µg/µl x 20 µl = 0.03 µg = 30 ng - Does this reaction fall within the optimal range of 1–10 µg/ml?
(50 ng vector + 30 ng insert) ÷ 20 µl = 4 µg/ml
|
|
 |
 |
|
New
England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201 |
|
 |
|
|
