Rules for good cloning
10/27/06
About vectors
1. Always have a map of your vector. Know the cloning sites, the size of the vector, the
sequencing primers that flank the MCS, and the antibiotic resistance.
2. Cut the vector with two enzymes if possible to allow for directional cloning. Stickyend sites are always better than blunt if you can do it.
3. Start with 2-4ug of the vector.
4. You begin by setting up two digests if you are using two enzymes, one for each of the
enzymes. Generally cut for 2-4 hours at 37 if you have the time to ensure that the vector
is optimally cut. If time is short, minimum time is 1 hour.
5. When cutting with two enzymes (for example EcoRI and XhoI) you want to know that
both enzymes cut. You also want to generate a positive control to test the efficiency of
your ligase. Therefore make a 1% gel and run all of one of the digests in one lane and
1/10th of the other digest in the other lane. You should see a single band in each lane.
That means both enzymes worked. You can then use the Qiagen gel extraction kit to
purify the band where you loaded the full digest and this will serve as your positive
ligation control.
6. You are now ready to cut the vector with the second enzyme. Say you purified the
whole EcoRI digest and tested 1/10th of the XhoI digest in the step above. Now take the
rest of the XhoI digest and purify it on a Qiagen gel extraction column WITHOUT
running it on a gel first. There is a protocol for this in the Qiagen book. Not using a gel
will help to limit your loss of DNA. Elute the DNA in 30uL then add the other enzyme
(EcoRI here) and buffer and make up to 40uL. Cut again.
7. Next phosphatase the vector to prevent it from religating with itself.When the digest is
finished, BEFORE running it on the gel, add 1uL SAP and 1uL SAP buffer and 8uL
ddH2O to phosphatase the vector for 15’ (for sticky end cuts) or 60’ (blunt ends) at 37
degrees. Heat the reaction to 65 degrees for 15’ to heat kill the enzyme. Technically if
you cut with two enzymes you don’t need this step, but it always helps to lower the
background. If you cut with only one enzyme you MUST include this step or your vector
background will be huge. Now you can gel purify the vector. Note if the vector is large,
adding 1 volume isopropanol to the melted gel band before loading it on the column will
improve your yield.
8. Finally, after gel purifying the vector, run 1/10 (3uL) of the eluted vector on a gel to
verify the size of the band, to ensure that no uncut vector is visible, and to estimate the
quantity (compare the brightness of the band to the brightness of the molecular weight
markers which are at a known concentration). Also take 2uL of the vector and get a
spectrophotometer reading. You need 50-100ng of the vector for the ligation, which
should be 2-5uL of the purified vector if all has gone well.
About inserts