Conversion of plasmids into Gateway compatible cloning
Rafael Martinez 14072011
Overview:
1. Select the right Gateway cassette (A, B or C).
2. Design primers to amplify the right Gateway cassette from a pDEST.
3. Open your plasmid with the enzyme of choice.
4. Create blunt ends in your plasmid by T4 DNA polymerase treatment.
5. To avoid self-ligation treat the plasmid with CIP.
6. Amplify by PCR the Gateway cassette of choice.
7. Perform a T4_kinase treatment.
8. Ligate the Gateway cassette with the open plasmid by T4 ligase.
9. Transform with ccdB SurvivalTM 2 T1R competent Cells
10. Analyze the transformants
Example:
In the following example we are going to convert a plasmid with a C-terminal tag into gateway
compatible cloning.
1. Selection of right Gateway Cassette and Enzyme to open the plasmid.
Select the right Gateway cassette (A, B or C) according with the position of the tag in your plasmid (at
N- or C-terminal). Also verify what enzyme will be the best to open the plasmid. Check the Gateway
Convertion Kit manual for more details.
In this example the Gateway cassette B is ideal because it will be in frame with a C-terminal tag from
the plasmid. For this example the plasmid needs to be open with BamIII.
2. Design primers to amplify the right Gateway cassette from a pDEST.
Do not add extra bases at the ends. Alternatively you can get the Gateway Conversion kit (Invitrogen
11828-029)
Primer
Forward primer
Reverse primer
Sequence
ATCAACAAGTTTGTACAAAAAAGC
ATCAACCACTTTGTACAAGAAAGCTG
Tm
60
65
3. Opening the plasmid.
In this example we digest the plasmid with BamHI. It is important to start with at least 4 µg of plasmid
since the amount will decrease due cleaning steps.
Opening plasmid
plasmid (144 ng/µl)
NEB Buffer 3 10x
BamHI
H20 (BSA 100µg/ml)
Total
Reaction
30
5
1
14
50
Amount
4 µg
20U
Clean the reaction with spin-column purification (Qiagen).
4. Create blunt ends in your plasmid by T4 DNA polymerase treatment.
If the enzyme digestion does not generate blunt ends, then is necessary to create them with a treatment
with T4 DNA Polymerase (NEB M023S). In this example the reaction is:
Blunt ends with T4 DNA polymerase
Digested plasmid (86ng/ml)
T4 DNA polymerase
dNTPs [10mM]
Buffer 2 10x
H20 (BSA 100µg/ml)
Total
Incubate 12°C for 15 min
Inactivate 75°C for 20 min.
Reaction
30
1
1
5
13
50
Concentration
2580
1U/µg
5. To avoid self-ligation treat the plasmid with CIP.
We used Alkaline Phosphatase, Calf Intestinal (CIP) (Finnzymes F-201S). CIP catalyses the removal of
5' phosphate residues from DNA to prevent self ligation. The manual of CIP recommends to calculate 1
pmol (1µM) of plasmid, in this example the plasmid has a size of 4026pb and according with the
following formula:
pmoles (µM) x length x 650 /106 = ng
1 pmol x 4026 x 650/ 106 = ng
1 pmol = 2616 ng
The CIP treatment is as the following:
CIP treatment
Plasmid (open+T4 treated) (86ng/ml)
CIP
Buffer 10X
Water
Total
Incubate 37°C for 1 hr
Inactivate 65°C for 15 min
50
0.5
5
X
50
2.6µg = 1pmol
Although the CIP manual does not comment about heat inactivation, this step is added in order to avoid
another spin-column cleaning (and reduce the amount of plasmid).
6. Amplify by PCR the Gateway cassette of choice.
Amplify by PCR the gateway cassette from a pDEST with primers designed in step 2. Use a High
Fidelity DNA Polymerase that generates blunt ends i.e Phusion (Finnzymes F-530S).
PCR Master mix
Buffer HF 5x
Forward primer (10µM)
Reverse primer (10µM)
dNTPs [10mM]
pDEST (200 ng/µl)
Phusion enzyme 2 U/µl
H2O
Total
1x 4X
10 40
1
4
1
4
1
4
0.5 2
1
4
35.5 142
50 200
PCR conditions
Temperature
Time
98°C
30s
94°C
10s
60°C
30s
72°C
30s
72°C
7 min
4°C
1 min
Cycles
1
30
1
1
PCR from Cassette B using pcDNA6.2 N-eGFP as a
template. The PCR product is 1713 pb
Run the PCR product in an agarose gel, cut the right band and clean it with a spin column purification
(i.e. Qiagen).
7. Perform a T4_kinase treatment with the PCR product
To clone blunt-end fragments is necessary that the insert has a 5' phosphate residues, normal PCR
primers are provided without them, if that is the case a step with a T4_kinase is needed to add them. At
this stage the plasmid does not have 5' phosphates since they were eliminated with the CIP treatment in
step 5. An example of the T4_kinase (NEB M0201S) is as the following:
T4_kinase treatment
Reaction
Gateway cassette 17ng/µl
44
T4_kinase Buffer 10x
5
T4_kinase 10U/µl
1
Total
50
Incubate 30 min at 37°C
Inactivate enzyme 65°C 20 min
8. Ligate the Gateway cassette with the open plasmid using T4 ligase.
Now is time to join the Gateway cassette and the plasmid. Try molar ratios of 1:1 and 1:3
(insert/vector). Here is a Promega tool that helps with that calculations. The following is an example of
a typical T4 ligase (NEB M0202S) reaction:
T4 DNA ligase reaction
Gateway cassette B_kinase treated 17ng/µl
Plasmid (open/T4/CIP/treated) 40ng/µl
Buffer 10x
T4 DNA ligase
H2O
total
Incubate overnight 20°C
Inactivate 65°C for 10 minutes
1
2.2
2.5
1
1
3.3
10
2
3.3
1.2
1
1
3.5
10
Tube1
Tube2
Ratio 1:1 Ratio 1:3
37.5ng
56.3ng
100ng
50ng
To be in the safe side leave the incubation overnight and inactivate with heat.
9. Transformation
Now the plasmid contains the Gateway cassette and therefore it needs to be cloned in competent cells
that are resistant to ccdB gene. Take 2µl of the ligation and transform in ccdB SurvivalTM 2 T1R
competent Cells (Invitrogen A10460) follow the protocol. Be sure to plate the cells on a LB-agar dish
with a proper antibiotic (resistance from the original plasmid) plus 30µg/ml of Cloramphenicol.
10. Analyze the transformants
Since a blunt-end ligation was done then 50% percent of the colonies will have the insert in the right
orientation and in the other 50% the insert will be inverted. Select several colonies and analyze them by
enzyme digestion.
Digestion analysis with SphI and BamHI of a plasmid converted to gateway compatible cloning. According with the expected
digestion pattern samples on wells 7-10 and 13 have the right construct. Samples in wells 1,2,4,6,11,12,14-16 have the gateway
cassette inverted.
Sequence the positive clones to verify that the Gateway cassette is in frame with the C-terminal tag.
Design primers at about 50 bp from the join insert/plasmid:
For plasmids with a C-terminal tag the triplet AAA located at the att2 sequence (gateway cassette) must be in frame with the start codon of
the C-terminal tag.
References
http://tools.invitrogen.com/content/sfs/manuals/gatewayvectorconversion_ccdbsurvival2_man.pdf
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cloning_guide.asp