Cla I - FAQs

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.

Relative enzyme activities can vary widely, and the amount used may also need to be adjusted according to the size and surrounding sequence of your target molecule. But to give a general idea of the relative activity of various enzymes, we performed a standardized digestion experiment using all of the enzymes listed below. The list indicates the number of units of enzyme that were needed for complete digest of 1 ?g supercoiled pBR322 DNA in 1 hour.

Enzyme - Units

Acc I - 4
Afl III - 0.5
Alu I - 1
AlwN I - 0.5
Ava I - 1
Ava II - 0.5 (a)
BstY I - 8
BamH I - 2
Ban II - 2
Bgl I - 8
Bsm I - 8
Cfo I - 4
Eco47 III - 8
Cla I - 1 (b)
Dde I - 1
Dra I - 4
EcoO109 I - 8
EcoR I - 0.5
EcoR II - >8 (a)
EcoRV - 2
Fsp I - 1
Hae II - 2
Hae III - 2
Hha I - 2
Hinc II - 4
Hind III - 0.5
Hinf I - 2
Hpa II - 1
Kpn2 I - 4
Mbo I - >8 (b)
Mbo II - 4 (b)
Msc I - >8 (a)
Mse I - 2
Msp I - 0.5
Nar I - 2
Nci I - 4
Nde I - 4
Nde II - >8 (b)
NgoA IV - 4
Nhe I - 2
Nsp I - 2
Nru I - 2 (b)
Psp5 II - 8
Pst I - 4
Pvu I - 2
Pvu II - 2
Rca I - 4
Rsa I - 4
Sal I - 8
Sau3A I - 2
Sau96 I - 8 (a)
Sca I - 4
Sph I - 2
Ssp I - 4
Sty I - 8
Taq I - 2 (b)
Tha I - 4
Vsp I - 0.5
Xma III - >8

(a) Sensitive to dcm methylation
(b) Sensitive to dam methylation

Notes:
- This list includes only restriction endonucleases with cleavage sites in pBR322, and not all enzymes listed are currently available for purchase from Thermo Fisher Scientific.
- In the experiments that generated this data, plasmid pBR322 was grown in E. coli RR1 and thus contained 5-methylcytosine and N6-methyladenine. Reactions contained 0.5, 1.0, 2.0, 4.0, or 8.0 units of enzyme, 1 µg of pBR322 DNA and the appropriate REACT Buffer in 20 µl. After a 1-h reaction, products were analyzed by electrophoresis on a 1% agarose gel.

1. Order of reagent addition may have been incorrect. Be sure to mix DNA with PLUS Reagent in dilution medium first, then combine this mixture with diluted Lipofectamine Reagent.

2. Diluted DNA or PLUS Reagent was not mixed well before pre-complexing. Be sure to mix DNA well when diluting before adding PLUS Reagent or vice versa. The order of addition of DNA and PLUS Reagent to the dilution medium for pre-complexing has no effect on the transfection activity.

3. No Lipofectamine Reagent was used. Ensure that DNA-PLUS Reagent complex is mixed and incubated with diluted Lipofectamine Reagent before adding to cells.

Restriction endonucleases should generally be stored at -20°C in a non frost-free freezer. Enzymes should be kept on ice during use and returned to the -20°C freezer as quickly as possible.
However, please be sure to double-check the recommended storage conditions in your product manual or packaging for any special instructions.

Certain restriction enzymes are unable to recognize and cleave at their target sites if specific adenine or cytosine residues in the sequence are methylated, and Dam and Dcm are two E. coli methylases which introduce methyl groups that affect the cutting sites of many common enzymes. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group from S-adenosylmethionine to the N6 position of the adenine residues in the sequence GATC. The Dcm methylase (encoded by the dcm gene; referred to as the Mec methylase in earlier references) methylates the internal cytosine residues in the sequences CCAGG and CCTGG at the C5 position.

To take advantage of Dam- and Dcm-sensitive restriction enzymes and get proper cleavage, plasmid DNA must be propagated in and isolated from an E. coli strain that is deficient in the endogenous Dam methylase and Dcm methylase enzymes just prior to the restriction reaction. We have one competent cell product available that is made with a dam- and dcm- strain: One Shot INV110 Chemically Competent E. coli (Cat. No. C7171-03).

Here are some recommendations:
1. You can verify the restriction endonuclease has activity by digesting the unit substrate (i.e., lambda or Ad-2 DNA) using the reaction conditions identified for unit definition: one unit of the restriction endonuclease should digest one µg of the unit substrate in one hour in the appropriate reaction buffer at the appropriate temperature.
2. You can test for the presence of inhibitors of restriction digestion in the sample DNA. Perform a restriction digest in which some of the sample DNA is included along with some of the unit substrate (i.e., lambda or Ad-2 DNA). If digestion of the unit substrate occurs alone but is not observed when the sample DNA is added, then a diffusible inhibitor of restriction digestion is present in the sample DNA. If digestion of the unit substrate occurs in the presence of the sample DNA, but the sample DNA is not digested, then the failure of the restriction endonuclease may be due to sensitivity of the restriction endonuclease to methylation in the sample DNA.
3. Finally, to identify any tube-specific issues like shipment or storage stability problems, you can test function by performing a side-by-side reaction with a different lot of the same Invitrogen restriction endonuclease and comparing results.

Three major sources of problems are described below:

1. The DNA isn't clean enough to cut.  Contaminating by-products from cells and purification chemicals can inhibit restriction enzymes. We recommend using high purity DNA isolation method like those found in our Purelink plasmid isolation products, or an equivalent protocol such as two phenol-chloroform extractions with isopropanol precipitations in between. See below for a suggested protocol.

2. The DNA may be heavily methylated. This can be checked by cutting with 4-base enzymes like HhaI, MboI, HpaII, etc. They should cut something - if they do not, that site is methylated. You can also check by cloning a piece of DNA and seeing if it now has the sites that the genomic DNA did not have.

3. The DNA may be very high in GC or very high in AT, and either extreme results in very little cutting by enzymes like EcoRI and HindIII. It is hard to tell the difference between very little cutting and no cutting with genomic DNA. This also can be tested by cutting with 4-base enzymes like HpaII (CCGG), HhaI (GCGC), MseI (TTAA) and DraI (TTTAAA). It might be better to do partial cuts with a 4-base cutter that will cut many times than to try to cut with a 6-base enzyme with more rare sites. Match enzymes and cloning sites, e.g. HpaII/ClaI, MseI/EcoRI, Sau3aI/BamHI.

Suggested protocol to purify plasmid from 200-400 mg cells:
1) Resuspend cells in 10 ml TE-lysozyme
2) Incubate 5 min at RT
3) Add 50 ml 10 mg/ml Proteinase K
4) Add 10 ml 1% SDS in TE
5) Vortex for 30 sec
6) Place at 45°C for 30 min or until clear
7) Vortex for 10 sec Can store overnight
8) Add 2 ml 3 N NaOAc
9) Add 20 ml phenol-CHCl3
10) Vortex 10 sec
11) Spin 20 min @ 3000-4000 RPM
12) Carefully remove up to 15 ml of supernatant (do not take milky interface) and place in 50 ml conical tube
13) Add 9 ml isopropanol
14) Invert several times to mix. Do not vortex. Precipitate should form wad.
15) Centrifuge 5 min @ 3000-4000 RPM or pick out with pipet
16) Wash pellet in 5 ml 70% EtOH
17) Resuspend pellet in 10 ml TE + 10 mg/ml RNAseA - place in 2 polypropylene 15ml conical tubes for 5 min 60 C
18) Add 0.5 ml 3 N NaOAc to each tube
19) Vortex 10 sec
20) Add 5 ml phenol/chloroform to each tube
21) Vortex 10 sec
22) Centrifuge 20 min @ 3000-4000 RPM
23) Remove 4 ml supernatant from each tube and place in 15 ml conical tube
24) Add 4.8 ml EtOH
25) Invert several times to mix. Precipitate should be a smaller single wad
26) Centrifuge or pick out precipitate
27) Wash in 1 ml 70% EtOH
28) Resuspend in 1 ml TE. Thick, very viscous, gel-like: 1 mg/ml. Dilute 2X. Very viscous, but clear solution: about 500 mg/ml. Slightly viscous, detectable when hand vortexing: about 100 mg/ml.
29) Place in tube marked with date. Store at -20°C for temporary storage, and -80°C for long term.

The product information sheet for an individual restriction enzyme will indicate any sensitivity it may have to methylation patterns. One Shot INV110 competent cells (Cat. No. C7171-03) are dam- and dcm-. Plasmid DNA isolated from these cells is not methylated by Dam methylase or Dcm methylase and can be digested with restriction enzymes that are sensitive to such methylation.

Please use our DoubleDigest Calculator linked below: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html

First, confirm the quality of your starting sample. Organic molecules and PCR inhibitors may be present in your RNA or DNA preparation, and these can affect amplification. In addition, it is unlikely that a single reaction mix and cycling program will work for every sample. You may need to optimize the conditions for the samples that failed. Parameters you can alter in your troubleshooting include primers, dNTPs, Mg, the amount of sample you are adding, and cycling conditions.