Extensions for LIC

Extensions for LIC

MHH modified 5/09

LIGATION INDEPENDENT CLONING FOR GENE TAGGING

(See Stols et al., 2002 Protein Expression and Purification 25;8-15)

Extensions for LIC primers

Coding strand Primer: 5’-TACTTCCAATCCAATTTAGC[add gene specific sequence here]

- Alternative to adding the GC at the end, the homology region of the gene-of-interest can be chosen to begin with a GC.

NOTE: It is essential to amplify a genomic gene fragment that includes a unique restriction site in order to linearize the construct prior to transfection. A 1kb fragment is sufficient for targeting, but it may be necessary to amplify a longer fragment so that it contains a unique site. The site should be at least 350 bp from either end of the fragment.

Non-coding strand primer: 5’-TCCTCCACTTCCAATTTTAGC[add gene specific sequence here]

- the gene is amplified up to the second last codon (i.e., does not include the terminator codon) to allow read-through to the reporter gene (e.g,. YFP, mCherry, etc).

* See schematic below for an example.

T4 Processing Protocols

Vector

  1. PacI digest LIC vector
  2. Use 5units/ug DNA, incubate 2-3 hours or O/N at 37oC. Phenol:chloroform extract and ethanol precipitate. Run small amount on gel to check for complete linearization. If linearization is incomplete then you will get substantial background.
  3. T4 Reaction
  4. Use roughly 1.6-2.0 ug linear DNA
  5. 60 ul reaction
  6. 12ul 5X buffer
  7. 3ul 100mM DTT
  8. 2.4ul 100mM dGTP
  9. 1.5ul T4 DNA polymerase (Novagen LIC qualified…Invitrogen is fine)
  10. 35.1ul dH20
  11. 6ul Template (200-300 ng/ul)
  12. Mix reaction on ice – add T4 pol last, mix gently in PCR machine
  13. Incubate 30 minutes at 22oC
  14. Shift to 75oC for 20 minutes
  15. Reduce temp. to 4oC
  16. Store in freezer until needed

Inserts

  1. Create fragment by PCR. Clean with Qiagen gel-extraction kit. Check DNA

Concentration.

  1. T4 Reaction
  2. Use roughly 0.2 pmol insert
  3. 20 ul reaction
  4. 4ul 5X buffer
  5. 1ul 100mM DTT
  6. 0.8ul 100mM dCTP
  7. 0.5ul T4 DNA polymerase (Novagen LIC qualified)
  8. 8.7 ul dH20
  9. 5ul Template
  10. Mix reaction on ice – add T4 pol last, mix gently in PCR machine
  11. Incubate 30 minutes at 22oC
  12. Shift to 75oC for 20 minutes
  13. Reduce temp. to 4oC
  1. Store in freezer until needed

Annealing

  1. Mix on ice
  2. 1ul of treated vector
  3. 2ul treated insert
  4. Incubate 10 minutes at RT
  5. Add 1 ul 25 mM EDTA, incubate 5 minutes at RT.
  6. Reduce temp. to 4oC
  7. Hold on ice until use. Use 1ul to transform cells.

Note: It is best to transform cells immediately after this reaction. However, the annealing reactions can be frozen and used again later. If you freeze them, thaw on ice and transform. Do not let them warm to room temperature. The same treated vector was frozen and used for several months without problems.

Transfection into Ku80KO parasites:

-Linearize 15ug construct, check on gel, phenol:chloroform extract, and ethanol precipitate.

-standard transfection protocol, 1x107 parasites/transfection, inoculate T25.

-Add drug the next day and keep under selection.

-Parasites lyse in 2 days, but the following passage generally crashes and takes 5-6 days to come back up.

-If using e.g., YFP-DHFR tagging vector, you should expect to see YFP positive parasite emerging after pass 3 or 4. In some cases the population may become homogeneously positive over time, but in other cases a mixed population may result necessitating screening of several clones. We have seen some evidence of this vector integrating at the DHFR locus, which may be responsible for some or all of the YFP negative parasites in the pyrimethamine resistant population.

YFP.LIC.DHFR vector: gatcggtaccCTGTACTTCCAATCCaatTTAATTAAAattGGAAGTGGAGGACGGgaattcATGGTGAGCAAGGGCGAGGA

PacI digest and T4 DNA Polymerase + dGTP treatment:

GATCGGTACCCTG T AAA ATT GGA AGT GGA GGA CGG gaattc (YFP)ATGGTGAGCAAGGGCGAGGA

CTAGCCATGGGACATGAAGGTTAGGTTAAAT GCC cttaag TACCACTCGTTCCCGCTCCT

Example :ACP gene 5’ end with LIC sequences TACTTCCAATCCAATTTAGCagacgatttatcctt…..

CGtctgctaaataggaa …..

ACP gene 3’ end with LIC sequences…….. GCT ACA GCG gc

…….. CGA TGT CGC cgA TTT TAA CCT TCA CCT CCT

* “Ligation” of insert with vector using complementary sequences on both:

GGTACCCTGTACTTCCAATCCAATTTAGCagacgatttatcctt………… GCT ACA GCC gcT AAA ATT GGA AGT GGA GGA CGG gaattc (YFP)ATGGTGA

CCATGGGACATGAAGGTTAGGTTAAATCGtctgctaaataggaa………. CGA TGT CGG cgA TTT TAA CCT TCA CCT CCT GCC cttaag TACCACTCGT

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