Directional Sub-Cloning Of DNA (one Common Restriction Site) Protocol

Overview

The process of DNA cloning is a critical component to target gene amplification. The protocol presented here offers a method for directional sub-cloning of DNA fragments that possess only one common restriction enzyme site (e.g. insert EcoRI and HindIII, receiving vector EcoRI and BamHI). This method provides increased flexibility for sub-cloning DNA fragments when common restriction enzymes sites are not available. DNA fragments are cloned using the common restriction site (EcoRI) and blunt ends generated using the Klenow fragment of DNA Polymerase I. The protocol contains steps for restriction digestion and Klenow treatment of both the vector containing the insert and the receiving vector, as well as, DNA digestion and storage.

Procedure

Note: Will generate blunt ends for cloning before digesting the DNA fragments with the common restriction enzyme.

1. Combine reaction components for Mix 1 to digest vector containing fragment. (see solutions list)
2. Digest DNA for at least 1.5 h at 37C in an incubator or PCR machine with heated lid. Avoid digesting DNA in a heat block since condensation inside the tube can change the reaction conditions.
3. Follow the previous two steps to perform the digestion of the receiving vector (using Mix 2).
4. If possible, heat inactivate enzymes following digestion by incubating digestions for 20 minutes at 65C (for this example, BamHI cannot be heat inactivated)
5. Fill-in 5’ overhangs to generate blunt ends (using Mix 3). Incubate reaction 15 minutes at 25C. To stop reaction, add EDTA (final concentration 10 mM) and incubate 20 minutes at 75C.
6. Add 2 ul of 10X DNA loading buffer and run reactions on a DNA agarose gel. If possible, load one entire reaction into one lane of the gel. Run gel at 100 – 110 volts (DNA is negatively charged, so it runs towards the positive pole) until DNA is sufficiently separated.
7. Confirm restriction digests are correct by comparing the banding pattern of the fragments on the gel with the expected sizes of the DNA fragments that should be obtained. Assuming the restriction enzymes used in steps 1 and 3 cut the respective vectors only once, both reactions should have linear bands that correspond to the size of the vectors.
8. Excise the desired band from the lane containing the insert using a clean scalpel or razor blade and place it in an epptube for gel extraction. Repeat to remove the desired band from the lane containing the receiving vector as well and place it in a different epptube. Attempt to excise as little agarose as possible while still removing the entire DNA fragment. If possible, 2 ml epptubes work well for this part of the protocol.
9. Gel extract DNA from agarose gel slices using a gel extraction kit. Elute DNA in as small of volume as possible, typically 10 ul H2O depending on which kit is used.
10. Combine reaction components for Mix 4 to digest the previously digested and Klenow treated DNA fragments with the common restriction enzyme (EcoRI).
11. Digest DNA for at least 1.5 h at 37C (repeat step 2).
12. Add 2 ul of 10X DNA loading buffer and run reactions on a DNA agarose gel. If possible, load one entire reaction into one lane of the gel. Run gel at 100 – 110 volts.
13. Confirm restriction digests are correct by comparing the banding pattern of the fragments on the gel with the expected sizes of the DNA fragments that should be obtained.
14. Excise the desired band from the lane containing the insert using a clean scalpel or razor blade. Repeat to remove the desired band from the lane containing the receiving vector as well. Attempt to excise as little agarose as possible while still removing the entire DNA fragment. Combine the insert and vector gel slices together in one epptube for gel extraction. If possible, 2 ml epptubes work well for this part of the protocol.
15. Gel extract DNA from agarose gel slices using a gel extraction kit. Elute DNA in as small of volume as possible, typically 10 ul H2O depending on which kit is used.
16. Ligate DNA fragments from gel extraction. Add ligase and ligase buffer directly to DNA eluted from gel extraction kit. If 10 ul is used for elution, usually 8-10 ul is recovered (Mix 5; see solutions list).
17. DNA can be frozen and is ready for transformation into E.coli. With good competent cells (1x108), cloning should yield 100-500 colonies (1/10 of the transformation) and 1000-2000 colonies (9/10 of the transformation) when plated on LB plates with the appropriate antibiotic selection.

Tips

1. If a site for a restriction enzyme that generates blunt ends is already present in one of the vectors and can be used for the cloning, then treatment with Klenow is not needed for that fragment.
2. Do not exceed more than 10% of the total volume of the reaction with restriction enzymes (1 ul of enzyme total per 10 ul of reaction). When using enzymes you are not familiar with, confirm the enzymes are active at 37C and not something higher or lower. If needed, adjust the temperature accordingly.
3. Klenow (with 3'→ 5' exonuclease activity) can be used to either fill-in a 5’ overhang or remove a 3’ overhang to generate a blunt end (for HindIII and BamHI, Klenow will fill the 5’ overhang). For both procedures, dNTPs must be added to the reaction for successful generation of blunt ends. It is best to add the dNTPs before addition of the enzyme.
4. For most fragments, a 0.8 - 1% agarose gel works well, for smaller fragments (less than 250 -500 bp) increase the percentage of agarose as needed. Add Ethidium Bromide to the gel directly before pouring to facilitate visualization of the DNA after running the gel.
5. Be sure to wear appropriate protection when reviewing DNA under UV light.
6. Ligation O/N at 4C (in refrigerator) works well and does not require a PCR machine for 16C. Ligation 2-4 hours at RT works, as well.
If desired, restriction digests or gel fragments can be stored at -20C, indefinitely. Before performing double digestions, it is best to confirm the restriction enzymes you are using are still active by performing single cut controls. For these controls, always use the same amount of enzyme and the reaction buffer that will be used for the double digestion.