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Xi-cloning is a very cheap and
efficient way of making directional blunt-end cloning. The only
requirements for using the techniques is that the recipient vector has at
least one unique restriction site and that 30 bp sequence on either side of
this restriction is known.
The techniques is described in US patent: US
6,936,470 B2 (Aug. 30, 2005) Liang et al. "RAPID AND ENZYMELESS CLONING OF
NUCLEIC ACID FRAGMENTS" which can be found by a simple Google search.
I have used the technique for two step construction
of replacement vectors for targeted gene replacement
in fungi (see Frandsen et al. 2006). However, the possible applications of
the technique is much wider and the ability to perform directional blunt-end
cloning makes it superior to standard restriction enzyme and ligase
dependent cloning. The technique is believed to be dependent on in vivo
homologous recombination between identical double stranded ends of the
insert and vector.
Materials needed:
 | Purified vector (pAg1-H3)
|
| Unique cutting restriction enzyme (best with
blunt cutting enzyme) |
| DNA phosphatase
|
| Klenow DNA polymerase or T4 DNA polymerase
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| Primers for the insert you
want to insert into the vector (the primers should be designed with 30 bp
long 5' overhangs identical to the sequences surrounding the restriction
enzyme site used to open the vector)
|
Protocol
Vector
- Make
three 10 ml LB + Kan (25
µg/ml)
cultures, and incubate
over night at 37 oC with 150-200 rpm.
-
Purify pAg1-H3 using QIAgen Miniprep kit (Velution = 35
µl
pr. purification, heat the tubes to 60 oC for 5 minutes before
the last spin for maximum extraction)
-
Digest the purified vector for 6 hours:
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Enzyme
|
3
µl
|
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Vector |
61
µl |
|
|
BSA |
8
µl |
| |
Buffer
|
8
µl |
- (If you are using a
restriction enzyme that leaves sticky ends:
Polish the ends of the digested vector with Klenow DNA polymerase or T4
DNA polymerase)
-
Dephosphorylate the
digested vector for 1 hour:
| |
CIP |
1
µl |
| |
Vector digest |
79
µl |
| |
CIP buffer |
8.8
µl |
-
Purify the vector using the QIAgen PCR purification kit (Velution
= 35
µl
pr. purification, heat the tubes to 60 oC for 5 minutes before
the last spin for maximum extraction). Measure DNA concentration on the
NanoDrop.
Insert
-
Amplify the desired insert using Accuzyme DNA polymerase (or
similar proof-reading DNA polymerase) (Vtotal
= 100
µl).
The primers should include 30 bp 5' overhangs
identical to the sequences surrounding the used restriction site in the
vector.
-
Check the success of the PCR reaction by pooling the PCR products and
checking 10
µl
on a gel.
-
Dephosphorylate the
insert for 1 hour:
CIP 1
µl
PCR product
90
µl
CIP buffer
10
µl
-
Purify the vector using the QIAgen PCR purification kit (Velution
= 35
µl
pr. purification, heat the tubes to 60 oC for 5 minutes before
the last spin for maximum extraction). Measure DNA concentration on the
NanoDrop.
"Ligation"/Transformation
- Mix
50 ng purified vector with 150 ng purified PCR product in a cooled 1.5 ml
Eppendorf tube
- Add
30
µl
chemical competent JM109 E. coli cells and mix gently
-
Incubate for 30 minutes on ice, heat shock at 42 oC for 45
seconds, return to ice for 2 minutes, add 970
µl
SOC medium, transfer to 37 oC with stir
at 300 rpm for 1 ½ hour.
-
Plate onto selective plates and incubate over night.
- Check
the resulting colonies by PCR using the primers used to amplify the insert
- Check
the size of the vector by restriction enzyme digestion
-
Seqeunce the insert to validate its identity and direction
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