Restriction Enzyme Cloning Support - Getting Started
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We recommend selecting at least one restriction enzyme that will leave a sticky end to force the orientation of the insert.
You can have all of the below controls or select the one you consider the most appropriate to the problem you are facing:
- Transform the E. coli with circular plasmid to assess the competency of the cells (how well they are taking up DNA).
- Transform and plate the dephosphorylated vector. It will help you assess how well the dephosphorylation worked and what proportion of colonies in your ligation transformation plate could be false positives (re-ligated vector or background).
- Use T4 DNA igase to re-ligate your cut vector, or lambda DNA/Hind III marker. It will help you assess whether the ligase itself is working properly.
Dephosphorylating the vector to decrease background can be achieved with:
- Heat Inactivated Alkaline Phosphatase, or
- Calf Intestinal Alkalline Phosphatase (available as 1 unit/μL or 20 units/μL)
If you need to create blunt phosphorylated DNA ends (“polishing” the ends), you can use:
- DNA End Repair Mix or T4 DNA polymerase or Klenow Fragment (large fragment) of E. coli DNA polymerase to generate blunt ends due to their 5’ →3’ DNA polymerase activity (filling-in of 5’ overhangs) and 3’→5’ exonuclease activity (chewing back of 3’ overhangs).
- All Anza modifying enzymes are compatible with the Anza Buffer.
- Anza Alkaline Phosphatase i supplied with the Anza Buffer.
- The Anza T4 PNK Kit, Anza™ DNA End Repair Kit, and the Anza DNA Blunt End Kit include their own buffers which contain co-factors for full functionality of each. They all are compatible with the Anza Buffer.
- The Anza T4 DNA Ligase Master Mix comes with no additional buffer and is fully compatible with Anza Buffer.
Digestions in several PCR reaction buffers were tested and the Anza restriction enzymes fully digested DNA. However, not all commercially available buffers could be tested.
To obtain the best results for unpurified PCR products, we recommend the following reaction conditions:
- Up to 10 µL of PCR reaction mixture
- 1 µL of 10X Anza Buffer
- 1 µL of Anza restriction enzyme
- Up to 20 µL of H2O
- 37 degrees C, 15 min
If digested PCR product will be used for cloning, it is necessary to purify the PCR fragment prior to digestion to remove the active polymerase. Active thermophilic DNA polymerase will still be present in the PCR mixture if it is not removed by purification and may alter the ends of the cleaved DNA thereby reducing ligation efficiency.
Isoschizomers are restriction enzymes that have the same recognition sequence and the same specificity. The first restriction enzyme discovered is called the prototype and all subsequently identified enzymes that recognize that sequence are isoschizomers.
- A unit is defined as the amount of enzyme required to completely digest 1 µg of substrate DNA in 50 µL of the reaction mixture in 1 hour at 37 degrees C.
- Anza restriction enzymes and buffer have been formulated such that 1 µL of Anza restriction enzyme cleaves 1 µg of substrate DNA in 15 minutes in Anza Buffer.
Star activity is the nonspecific cleavage of sequences that are similar to a recognition sequence, but not identical to the recognition sequence. This occurs when reaction conditions are suboptimal. Some restriction enzymes have more of a propensity for star activity than others. Factors that are included in suboptimal conditions that result in star activity include high glycerol concentration, high concentration of enzyme, prolonged incubation time, and use of the incorrect buffer.
Anza restriction enzymes in conjunction with the Anza Buffer have been formulated to eliminate star activity when following the recommended protocol. Anza enzymes allow for the flexibility for complete digestion in as few as 15 minutes and longer reactions, up to 16 hours, can be safely digested without showing star activity.
Anza restriction enzymes contain a number preceding the name. This number provides the option to organize the enzymes in freezers numerically or alphabetically. More common enzymes tend to have a lower number.
When the next step following digestion is agarose gel electrophoresis, the Anza Red Buffer can be used directly. The Anza Red Buffer contains the necessary loading dye and density gradient for directly loading on to the agarose gel. The red dye does not interfere with digestion and helps reduce additional pipetting.
Use the clear Anza Buffer for applications that require analysis by fluorescence excitation, as the dyes in the Anza Red Buffer may interfere with some fluorescence measurements.
For optimal results, we highly recommend exclusively using Anza restriction enzymes in double digestion as they all have 100% buffer compatibility. If a certain enzyme is not available in Anza format, but is available in FastDigest format or as a Thermo Scientific conventional restriction enzyme, it may be possible to perform a double digestion. For specific protocol recommendations, please contact our technical support with detailed information of the desired enzymes and DNA template.
Enzymes may freeze during shipment on dry ice. This does not affect them as they have been tested and shown to be 100% active after three freeze-thaw cycles. For 24-48 hour delivery, enzymes may be shipped on ice pack as their performance is not affected by short exposure times at 4 degrees C.
The Anza Buffers can be stored at 4 degrees C for at least 6 months. To maintain effectiveness through the expiration date, we recommend longer term storage of both the Anza 10X Buffer and Anza 10X Red Buffer at -20 degrees C.
As an added convenience, BSA is included in the Anza 10X Buffers and Anza 10X Red Buffers. This eliminates the need to add BSA in a separate step.
No, the old enzymes and buffers are not compatible with the new enzymes and buffers.
In general, 1 unit of enzyme can digest 1 microgram of lambda DNA in 50 microliter solution in 1 hour at 37 degrees C. Please refer to the manual for specific temperature/time for your enzyme.
If you are able to find a buffer in which all 3 enzymes have sufficient activity (usually not lower than 50%), you can set up a single digestion with all 3 enzymes. It is important that the total volume of enzymes you add to your reaction is not more than 1/10th of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol exceeds 5%. If you are not able to find a buffer in which all your enzymes have sufficient activity, you will have to perform sequential digestions of the plasmid with the individual enzymes.
Make sure you have inactivated the ligase and store the ligation reaction at 4 degrees C.
Please consider the following suggestions:
- Try different molar ratios of insert to vector. Having an excess of insert is usually what will work, try 1:1 to 15:1 insert:vector.
- Try increasing the time of the ligation at 37 degrees C.
- Try performing the ligation at 16 degrees C overnight (you can set it up on your PCR machine).
dATP is a competitive inhibitor of T4 DNA ligase. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2 M) of Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.
The main difference between the 2 enzymes is that E. coli DNA Ligase cannot ligate blunt dsDNA fragments. Both ligases can be used to repair single stranded nicks in duplex DNA and to perform cohesive or sticky end ligations. E. coli DNA Ligase is generally used to seal nicks during second strand cDNA synthesis, since T4 DNA Ligase could result in formation of chimeric inserts.
ExpressLink™ T4 DNA Ligase is a T4 DNA ligase that catalyzes the ligation of blunt or cohesive-end DNA fragments in only 5 minutes at room temperatures or ligation of PCR fragments with A’ overhangs in 15 minutes. ExpressLink™ T4 DNA Ligase undergoes superior exonuclease quality control assays for exonuclease-free ligations. The enzyme is supplied at a concentration of 5 units/μL.
You may have to try different ratios from 1:1 to 15:1 insert:vector.
For Research Use Only. Not for use in diagnostic procedures.