Xi-cloning

14/07/10

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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:

bullet    Purified vector (pAg1-H3)
bullet    Unique cutting restriction enzyme (best with blunt cutting enzyme)
bullet    DNA phosphatase
bullet    Klenow DNA polymerase or T4 DNA polymerase
bullet    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

  1. Make three  10 ml LB + Kan (25 g/ml) cultures, and incubate over night at 37 oC with 150-200 rpm.
  1. Purify pAg1-H3 using QIAgen Miniprep kit (Velution = 35 l pr. purification, heat the tubes to 60 oC for 5 minutes before the last spin for maximum extraction)
  1. Digest the purified vector for 6 hours:
  Enzyme        3 l
  Vector 61 l
                 BSA 8
       Buffer        8 l

     

  1. (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)
  2. Dephosphorylate the digested vector for 1 hour:
  CIP 1 l
  Vector digest 79 l
  CIP buffer 8.8 l

 

  1. Purify the vector using the QIAgen PCR purification kit (Velution = 35 l 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

  1. Amplify the desired insert using Accuzyme DNA polymerase (or similar proof-reading DNA polymerase) (Vtotal = 100 l). The primers should include 30 bp 5' overhangs identical to the sequences surrounding the used restriction site in the vector.
  2. Check the success of the PCR reaction by pooling the PCR products and checking 10 l on a gel.
  3. Dephosphorylate the insert for 1 hour:

CIP                                     1 l

PCR product                        90 l

CIP buffer                           10 l

 

  1. Purify the vector using the QIAgen PCR purification kit (Velution = 35 l 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

  1. Mix 50 ng purified vector with 150 ng purified PCR product in a cooled 1.5 ml Eppendorf tube
  2. Add 30 l chemical competent JM109 E. coli cells and mix gently
  3. Incubate for 30 minutes on ice, heat shock at 42 oC for 45 seconds, return to ice for 2 minutes, add 970 l SOC medium, transfer to 37 oC with stir at 300 rpm for 1 hour.
  4. Plate onto selective plates and incubate over night. 
  5. Check the resulting colonies by PCR using the primers used to amplify the insert
  6. Check the size of the vector by restriction enzyme digestion
  7. Seqeunce the insert to validate its identity and direction

 

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Dette sted blev sidst opdateret 14. July 2010