Cloning genes using Cre-loxP - based on Steve Elledge’s paper http://signal.salk.edu/SSP/Elledgepaper.pdf

Transform E. coli, select for Kan resistant (KnR) colonies

GST-Cre protein purification protocol

1. In a 250 ml flask, use a 2-5 ml overnight culture to inoculate 50 ml of LB + AMP

(100ug/ml) media with E. coli strain BL21 containing the GST-Cre plasmid

pQL123; grow to an OD600 between 0.4-0.8 in at 37 C with shaking (~200 rpm).

2. Add IPTG to 0.3 mM. Continue growth at 25 C (or room temperature) overnight

(12-16 hours).

3. Harvest cells by spinning 3.5 Krpm for 5 minutes. Discard supernatant and place

cell pellet on ice. Keep cells/protein cold at all times from now on.

4. Resuspend cell pellet in 1.5 ml cold buffer L, freshly supplemented with protease

inhibitors (1X). Use a blue tip and pipettor to resuspend cells.

5. Sonicate cells 3 x 10 seconds on 20% power. Place cells on ice for 1 minute

between sonications. Avoid foaming the cell suspension by keeping the sonicator

tip below the surface of the solution and sonicating on low power.

6. Transfer 1.3 ml of lysed solution to a microfuge tube and spin at ~13,000 rpm for

5 minutes at 4 C (use prechilled refrigerated centrifuge in Cell lab – need to

change out the rotor beforehand and allow to cool to 4 C).

7. Transfer supernatant to a new tube and store on ice.

BAIT vectorPREY vector

8. GST-Cre protein binding : Add 200 ul of washed glutathione-beads (make sure to

resuspend the bead slurry prior to pipetting) to the supernatant and place on ice

for 30 to 60 minutes. Every 5-10 minutes, gently mix to keep beads in solution.

9. Gently pellet the beads by spinning at 2000 rpm for 30 seconds. Carefully remove

the supernatant using a pipettor.

10. Wash the beads 3 times : resuspend beads in 1ml of buffer W, spin as in step 9,

discarding supernatant each time.

11. Elutions : add 100 ul of buffer E, mix occasionally and gently, and let stand at RT

for 5 minutes. Spin as in step 9 to pellet beads; remove supernatant to new tube

and label “E1”. Repeat elution, saving supernatant as “E2”.

12. Save beads; label tube as “beads” and store beads and elutions at 4 C.

Buffer L: 20 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 or tween 20

detergent

Glutathione beads (50% slurry) in Buffer L

Buffer W: 100 mM Tris-Cl pH 8.0, 100 mM NaCl

Buffer E: Buffer W + 20 mM glutathione

pUNI 51 plasmid sequence1 cttcccaacc ttaccagagg gcgccccagc tggcaagcta gctgtccgaa atattataaa 61 ttatcgcaca cataaaaacc atgctgttgg tgtgtctatt aaatcgattt tttgttataa 121 cagacactgc ttgtccgata tttgatttag gatacaggta ccaattaacc ctcactaaag 181 ggataacttc gtatagcata cattatacga agttatctgg aattcggccg tcaaggccac 241 aggatcccta gagggcctca tgggccgtcg actagaattg tgagcgctca caattctagc 301 ggccgccgag atcatatcac tgtggacgtt gatgaaagaa tacgttattc tttcatcaaa 361 tcgtggtcga tcgacgagct cgctatcagc ctcgactgtg ccttctagtt gccagccatc 421 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 481 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 541 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 601 ggattctaga agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 661 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt 721 tgctgaaagg aggaactata tccggatatc atgcatcgcg agagagagag agagagagag 781 agagagagag agagagagag agacgtgggc ccaattctgt cagccgttaa gtgttcctgt

841 gtcactgaaa attgctttga gaggctctaa gggcttctca gtgcgttaca tccctggctt 901 gttgtccaca accgttaaac cttaaaagct ttaaaagcct tatatattct tttttttctt 961 ataaaactta aaaccttaga ggctatttaa gttgctgatt tatattaatt ttattgttca 1021 aacatgagag cttagtacgt gaaacatgag agcttagtac gttagccatg agagcttagt 1081 acgttagcca tgagggttta gttcgttaaa catgagagct tagtacgtta aacatgagag 1141 cttagtacgt gaaacatgag agcttagtac gtactatcaa caggttgaac tgctgatcaa 1201 cagatcctct acactagtct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 1261 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 1321 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 1381 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 1441 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 1501 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 1561 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 1621 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca 1681 ttcgccattc aggctgcacg cgtttcgaac cccagagtcc cgctcagaag aactcgtcaa 1741 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga 1801 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt 1861 cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat 1921 tttccaccat gatattcggc aagcaggcat cgccatgtgt cacgacgaga tcctcgccgt 1981 cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt 2041 cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc 2101 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca 2161 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct 2221 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca 2281 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca 2341 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg 2401 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga 2461 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc 2521 gaaacgatcc tcatcctgtc tcttgatcag atct