CLONING GUIDE WORKFLOW
RI GENOMICS & SEQUENCING CENTER
University of Rhode Island
352 CBLS - 120 Flagg Road
Kingston, RI 02881
Created by Ted Spinard, Kingston, RI, October 2013
1
Cloning guide flow chart:

Use PCR to amplify your fragment of interest. Run the product on an agarose gel.
o Is there one fragment?
 Yes
 Is it the correct size?
o Yes
 PCR column purify the product and proceed to your specific
Digestion
o No
 Tweak the parameters or redesign your primers
 No
 Have you tried tweaking the parameters?
o Yes
 Is there a strong band at the correct size
 Yes
o Gel extract it. Use this band as your
template and re-run the PCR.
 No
o Redesign your primers

Specific digestions
o Single digestion:
o Double digestion w/ compatible buffers (cut sites located <40bp apart):
o Double digestion w/ compatible buffers (cut sites located >40bp apart):
o Double digestion w/ non-compatible buffers (cut sites located <40bp apart):
o Double digestion w/ non-compatible buffers (cut sites located >40bp apart):
2
pg. 3
pg. 6
pg. 9
pg. 12
pg. 15

Control: Digest your plasmid with the enzyme and run 1000 ng of product on a gel
o Is there a single band?
 Yes
 Good. Digest your plasmid. Proceed to PCR column purify
 No
 Do the multiple bands correspond to the uncut plasmid?
o Yes
 Your enzyme efficiency is low. Try using a different buffer or
order a new enzyme
o No
 Your enzyme is cutting nonspecifically or
 Your enzyme is undergoing star activity. Recheck your
plasmid map and buffer compatibility.
 Or your enzyme is cutting at multiple sites
o PCR column purify:
 Clean your digested PCR product using a commercial PCR column purification kit.
Check the DNA concentration after the elution step to ensure you still have product.
 Clean your digested plasmid product using a commercial PCR column purification
kit. Check the DNA concentration after the elution step to ensure you still have
product.
 Proceed to CIAP
o CIAP:
 Note: Run the Transformation controls and the ligation controls first
 Control
 Treat the single cut purified plasmid with CIAP. PCR column purify. Run a
ligation and transform.
o Are there zero or very few colonies?
 Yes
 Good. The CIAP is working. Use the CIAP treated
single cut plasmid and proceed to the ligation
cloning step
 No
 Your CIAP is expired
o Ligation:
 Note: Run the Transformation controls 1st
 Positive control (Do not treat with CIAP)
 Treat the single cut purified plasmid with ligase. Transform and plate your
cells.
o Are there hundreds of colonies?
3




o
Yes

If the negative control also works…good your ligase
works. Run the CIAP control.


Your ligase may be expired
You may not be starting with a high enough DNA
concentration
No
Negative control
 Don’t treat the single cut and purified plasmid with ligase. Transform your
bacteria with this cut, untreated plasmid. Transform and plate your cells
o Are there few if any colonies?
 Yes
 If the positive control also works…good your ligase
works. Run the CIAP control.
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Your restriction enzymes are not cutting well
enough
Cloning:
 Combine your purified cut plasmid and purified cut PCR product and run a
ligation. Proceed to transformation cloning
Transformation:
 Positive control
 Transform your cells with uncut plasmid
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your cells
are competent. Run the ligation controls.
 No
 Your cells are no longer competent. Make new
competent cells
 Your plasmid is junk. Run on a gel to confirm it is
uncut
 Make sure you are using the correct plates
 Negative control
 Without transforming, plate your bacteria on the selective medium
o Are there zero colonies?
 Yes
 If the positive control also works…good your cells
are competent. Run the ligation controls
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Cloning:
4

Transform your competent cells with the ligated plasmid/insert. There is no
need to purify the ligation reaction, however you may wish to inactivate the
ligase by heating it to the specified inactivation temperature.
o Are there colonies?
 Yes
 Check to see if the colonies have the insert by using
any of the techniques below.
o Run a colony PCR using primers flanking the
insert site. Be sure to include a negative
control (cells containing a plasmid without
the insert). The PCR product should be the
size of the insert + the flanking region.
o Grow the colonies overnight and Miniprep
the cells to extract the plasmid. Run a PCR
using primers flanking the insert site. Be
sure to include a negative control (plasmid
without the insert). The PCR product should
be the size of the insert + the flanking
region.
o Grow the colonies overnight and Miniprep
the cells to extract the plasmid. Restriction
enzyme digest the plasmid with cut sites
located away from where the insert is
located. This should generate different
sized fragments compared to a plasmid
without the insert.
o Run uncut plasmid versus plasmid with
insert if the insert has a ≥ 200bp size shift.

No



Try again, make sure you follow the controls.
Make sure you are starting with enough DNA to
have enough DNA for the ligation step.
Vary insert:vector ratios in ligation.
5
o
Control: Digest your plasmid with each enzyme and run 1000 ng on a gel.
 Is there a single band?
 Yes
o Good. Proceed to Control 2
 No
o Do the multiple bands correspond to the uncut plasmid?
 Yes
 Your enzyme efficiency is low. Try using a different buffer or
order a new enzyme
 No
o Your enzyme is cutting nonspecifically or
o Your enzyme is undergoing star activity. Recheck your plasmid map
and buffer compatibility.
o Or your enzyme is cutting at multiple sites
o
Control 2: Digest your plasmid with the enzymes together and run 1000 ng of product on a gel
 Is there a single band?
 Yes
o Good. Proceed to PCR column purify
 No
 Your enzyme is cutting nonspecifically or is undergoing star activity. Recheck your
plasmid map and buffer compatibility.
o
o
PCR column purify:
 Clean your digested PCR product using a commercial PCR column purification kit. Check the
DNA concentration after the elution step to ensure you still have product.
 Clean your digested plasmid product using a commercial PCR column purification kit. Check
the DNA concentration after the elution step to ensure you still have product.
 Proceed to ligation
Ligation:
 Note: Run the Transformation controls 1st
 Positive control
 Treat the single cut purified cut plasmid with ligase. Transform and plate your cells
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your ligase works.
Proceed to ligase cloning
 No
 Your ligase may be expired
6



o
You may not be starting with a high enough DNA
concentration
Negative control
 Don’t treat the single cut plasmid with ligase. Transform with this plasmid and plate.
o Are there zero colonies?
 Yes
 If the positive control also works…good your ligase works. to
ligase cloning
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Your restriction enzymes are not cutting well enough
Cloning:
 Combine your purified cut plasmid and purified cut PCR product and run a ligation.
Proceed to transformation cloning
Transformation:
 Positive control
 Transform your cells with uncut plasmid
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your cells are
competent. Run the ligation controls.
 No
 Your cells are no longer competent. Make new competent
cells
 Your plasmid is junk. Run on a gel to confirm it is uncut
 Make sure you are using the correct plates
 Negative control
 Without transforming, plate your bacteria on the selective medium
o Are there zero colonies?
 Yes
 If the positive control also works…good your cells are
competent. Run the ligation controls
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Cloning:
 Transform your competent cells with the ligated plasmid/insert. There is no need to
purify the ligation reaction, however you may wish to inactivate the ligase by
heating it to the specified inactivation temperature.
o Are there colonies?
 Yes
 Check to see if the colonies have the insert by using any of
the techniques below.
o Run a colony PCR using primers flanking the insert
site. Be sure to include a negative control (cells
7
o
o

containing a plasmid without the insert) The PCR
product should be the size of the insert + the
flanking region.
Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Run a PCR using primers
flanking the insert site. Be sure to include a negative
control (plasmid without the insert) The PCR
product should be the size of the insert + the
flanking region.
Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Restriction enzyme digest
the plasmid with cut sites located away from where
the insert is located. This should generate different
sized fragments compared to a plasmid without the
insert.
No



Try again, make sure you follow the controls.
Make sure you are starting with enough DNA to have
enough DNA for the ligation step.
Vary insert:vector ratios in ligation.
8

Control: Digest your plasmid with each enzyme and run 1000 ng on an agarose gel
 Is there a single band?
 Yes
o Good. Proceed to Control 2
 No
o Do the multiple bands correspond to the uncut plasmid?
 Yes
 Your enzyme efficiency is low. Try using a different buffer or
order a new enzyme
 No
 Your enzyme is cutting nonspecifically or is undergoing star
activity. Recheck your plasmid map and buffer compatibility.

Control 2: Digest your plasmid with the enzymes together and run 1000 ng of product on a gel
 Is there a single band?
 Yes
o Good. If the fragment is below a certain size it will not show up on the gel.
Proceed to Gel purify
 No
o Do the multiple bands correspond to the correct sized fragments?
 Yes
 Good. Proceed to Gel purify
 No
 Your enzyme is cutting nonspecifically or is undergoing star
activity. Recheck your plasmid map and buffer compatibility.
Gel Purify:
o Follow a protocol to extract the correct sized plasmid fragment from your agarose gel.
Check the DNA concentration after the elution step to ensure you still have product. The
concentration will decrease dramatically. To increase your concentration, purify multiple
bands in the same column. Proceed to PCR column purification.
PCR column purify:
o Clean your PCR digested product using a commercial PCR column purification kit. There is no
need to clean your gel purified cut plasmid. Check the DNA concentration after the elution
step to ensure you still have product.



Ligation:
o Note: Run the Transformation controls 1st
o Positive control
9



o
Treat the single cut purified cut plasmid with ligase. Transform and plate your cells
 Are there hundreds of colonies?
o Yes
 If the negative control also works…good your ligase works.
Proceed to ligase cloning
o No
 Your ligase may be expired
 You may not be starting with a high enough DNA
concentration
Negative control
 Don’t treat the single cut plasmid with ligase. Transform with this plasmid and plate.
 Are there zero colonies?
 Yes
 If the positive control also works…good your ligase works.
Proceed to ligase cloning
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Your restriction enzymes are not cutting well enough
Cloning:
 Combine your purified cut plasmid and purified cut PCR product and run a ligation.
Proceed to transformation cloning
Transformation:
 Positive control
 Transform your cells with uncut plasmid
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your cells are
competent. Run the ligation controls.
 No
 Your cells are no longer competent. Make new competent
cells
 Your plasmid is junk. Run on a gel to confirm it is uncut
 Make sure you are using the correct plates
 Negative control
 Without transforming, plate your bacteria on the selective medium
o Are there zero colonies?
 Yes
 If the positive control also works…good your cells are
competent. Run the ligation controls
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Cloning:
10

Transform your competent cells with the ligated plasmid/insert. There is no need to
purify the ligation reaction, however you may wish to inactivate the ligase by
heating it to the specified inactivation temperature.
o Are there colonies?
 Yes
 Check to see if the colonies have the insert by using any of
the techniques below
o Run a colony PCR using primers flanking the insert
site. Be sure to include a negative control (cells
containing a plasmid without the insert). The PCR
product should be the size of the insert + the
flanking region.
o Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Run a PCR using primers
flanking the insert site. Be sure to include a negative
control (plasmid without the insert). The PCR
product should be the size of the insert + the
flanking region.
o Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Restriction enzyme digest
the plasmid with cut sites located away from where
the insert is located. This should generate different
sized fragments compared to a plasmid without the
insert.

No



Try again, make sure you follow the controls.
Make sure you are starting with enough DNA to have
enough DNA for the ligation step.
Vary insert:vector ratios in ligation.
11
o
Control: Digest your plasmid with each enzyme
 Is there a single band?
 Yes
o Good. Digest your PCR product. Proceed to PCR column purify
 No
o Do the multiple bands correspond to the uncut plasmid?
 Yes
 Your enzyme efficiency is low. Try using a different buffer or
order a new enzyme
 No
 Your enzyme is cutting nonspecifically or is undergoing star
activity. Recheck your plasmid map and buffer compatibility.



o
PCR column purify:
o Clean your digested PCR product using a commercial PCR column purification kit. Check the
DNA concentration after the elution step to ensure you still have product.
o Clean your digested plasmid product using a commercial PCR column purification kit. Check
the DNA concentration after the elution step to ensure you still have product.
 Proceed to second digestion
Second Digestion
o Digest your single cut PCR product
o Digest your single cut plasmid product
 Proceed to PCR column purify.
PCR column purify:
o Clean your digested PCR product using a commercial PCR column purification kit. Check the
DNA concentration after the elution step to ensure you still have product.
o Clean your digested plasmid product using a commercial PCR column purification kit. Check
the DNA concentration after the elution step to ensure you still have product.
 Proceed to ligation
Ligation:
 Note: Run the Transformation controls 1st
 Positive control
 Treat the single cut purified cut plasmid with ligase. Transform and plate your cells
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your ligase works.
Proceed to ligase cloning
 No
12




o
Your ligase may be expired
You may not be starting with a high enough DNA
concentration
Negative control
 Don’t treat the single cut plasmid with ligase. Transform with this plasmid and plate.
o Are there zero colonies?
 Yes
 If the positive control also works…good your ligase works. to
ligase cloning
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Your restriction enzymes are not cutting well enough
Cloning:
 Combine your purified cut plasmid and purified cut PCR product and run a ligation.
Proceed to transformation cloning
Transformation:
 Positive control
 Transform your cells with uncut plasmid
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your cells are
competent. Run the ligation controls.
 No
 Your cells are no longer competent. Make new competent
cells
 Your plasmid is junk. Run on a gel to confirm it is uncut
 Make sure you are using the correct plates
 Negative control
 Without transforming, plate your bacteria on the selective medium
o Are there zero colonies?
 Yes
 If the positive control also works…good your cells are
competent. Run the ligation controls
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Cloning:
 Transform your competent cells with the ligated plasmid/insert. There is no need to
purify the ligation reaction, however you may wish to inactivate the ligase by
heating it to the specified inactivation temperature.
o Are there colonies?
 Yes
 Check to see if the colonies have the insert by using any of
the techniques below.
13
o
o
o

Run a colony PCR using primers flanking the insert
site. Be sure to include a negative control (cells
containing a plasmid without the insert) The PCR
product should be the size of the insert + the
flanking region.
Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Run a PCR using primers
flanking the insert site. Be sure to include a negative
control (plasmid without the insert) The PCR
product should be the size of the insert + the
flanking region.
Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Restriction enzyme digest
the plasmid with cut sites located away from where
the insert is located. This should generate different
sized fragments compared to a plasmid without the
insert.
No



Try again, make sure you follow the controls.
Make sure you are starting with enough DNA to have
enough DNA for the ligation step.
Vary insert:vector ratios in ligation.
14
o
Control: Digest your plasmid with each enzyme
 Is there a single band?
 Yes
o Good. Digest your PCR product. Proceed to PCR column purify
 No
o Do the multiple bands correspond to the uncut plasmid?
 Yes
 Your enzyme efficiency is low. Try using a different buffer or
order a new enzyme
 No
 Your enzyme is cutting nonspecifically or is undergoing star
activity. Recheck your plasmid map and buffer compatibility.




o
PCR column purify:
o Clean your digested PCR product using a commercial PCR column purification kit. Check the
DNA concentration after the elution step to ensure you still have product.
o Clean your digested plasmid product using a commercial PCR column purification kit. Check
the DNA concentration after the elution step to ensure you still have product.
 Proceed to second digestion
Second Digestion
o Digest your single cut PCR product
 Proceed to PCR column purify #2:
o Digest your single cut plasmid product
 Proceed to Gel Purify for cut plasmid:
Gel Purify:
o Follow a protocol to extract the correct sized plasmid fragment from your agarose gel.
Check the DNA concentration after the elution step to ensure you still have product. The
concentration will decrease dramatically. To increase your concentration, purify multiple
bands in the same column. Proceed to PCR column purification.
PCR column purify:
o Clean your digested PCR product using a commercial PCR column purification kit. Check the
DNA concentration after the elution step to ensure you still have product.
 Proceed to ligation
Ligation:
 Note: Run the Transformation controls 1st
 Positive control
 Treat the single cut purified cut plasmid with ligase. Transform and plate your cells
o Are there hundreds of colonies?
 Yes
15



o

If the negative control also works…good your ligase works.
Proceed to ligase cloning


Your ligase may be expired
You may not be starting with a high enough DNA
concentration
No
Negative control
 Don’t treat the single cut plasmid with ligase. Transform with this plasmid and plate.
o Are there zero colonies?
 Yes
 If the positive control also works…good your ligase works. to
ligase cloning
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Your restriction enzymes are not cutting well enough
Cloning:
 Combine your purified cut plasmid and purified cut PCR product and run a ligation.
Proceed to transformation cloning
Transformation:
 Positive control
 Transform your cells with uncut plasmid
o Are there hundreds of colonies?
 Yes
 If the negative control also works…good your cells are
competent. Run the ligation controls.
 No
 Your cells are no longer competent. Make new competent
cells
 Your plasmid is junk. Run on a gel to confirm it is uncut
 Make sure you are using the correct plates
 Negative control
 Without transforming, plate your bacteria on the selective medium
o Are there zero colonies?
 Yes
 If the positive control also works…good your cells are
competent. Run the ligation controls
 No
 Your plates are expired
 Your bacteria have resistance to the antibiotic
 Cloning:
 Transform your competent cells with the ligated plasmid/insert. There is no need to
purify the ligation reaction, however you may wish to inactivate the ligase by
heating it to the specified inactivation temperature.
o Are there colonies?
 Yes
16


Check to see if the colonies have the insert by using any of
the techniques below
o Run a colony PCR using primers flanking the insert
site. Be sure to include a negative control (cells
containing a plasmid without the insert) The PCR
product should be the size of the insert + the
flanking region.
o Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Run a PCR using primers
flanking the insert site. Be sure to include a negative
control (plasmid without the insert) The PCR
product should be the size of the insert + the
flanking region.
o Grow the colonies overnight and Miniprep the cells
to extract the plasmid. Restriction enzyme digest
the plasmid with cut sites located away from where
the insert is located. This should generate different
sized fragments compared to a plasmid without the
insert.


Try again, make sure you follow the controls.
Make sure you are starting with enough DNA to have
enough DNA for the ligation step.
Vary insert:vector ratios in ligation.
No

17