Objective: Transfer aminoacyl RNA synthetases from thermophile genomic dna into non-thermophillic plasmids, and transform into DH10B cells
1. Isolate aminoacyl tRNA synthetases from thermophillic genomic DNA via PCR with designed oligos.
2. Digest target vector (pBad-myc/hisA) and tRNA synthetase pcr products.
3. Ligate each pcr product into vector
4. Transform each ligation reaction
5. Pick colonies, grow cultures, and miniprep DNA for sequencing
Thermophile: thermatoga maritma
tRNA synthetases: Ala, Asp, Cys, Lys, Leu, Ile, Glu, Phe, His, Gly
User:Cheryl He 12:22 13 February 2014 (EST)
organism: thermatoga maritima
synthetases: asp, cys, lys, leu, ile, glu, phe, his, gly
Cheryl He 13:28, 27 February 2014 (EST)
gel: 34mL ddh2O + 700 uL TAE buffer + 0.35 g agarose. microwave, cool, add 3.5 uL. run gel at voltage 160
miniprep: from 2yt culture, miniprep 1.5ul. from lb culture miniprep 2 ul.
1. spin down cells full speed ~45 s
2. pour out supernatant
3. add 250ul p1 buffer, vortex
4. add 250 ul p2 buffer
5. add 300 ul n3 invert
6. spin for 5 min
7. pour out lysate onto column, spin 15 s
8. add PB buffer 500ul, spin 15 s
9. add PE buffer 750 ul, spin 15 s
10. remove residual water/ethanol
11. use same tube, spin for 90s
12. elute in 50ul ddh2O
13. spin 30 s
Cheryl He 13:21, 6 March 2014 (EST)
march 4: Make 13 pcr products of tRNA-synthetases using expand buffer
24 ul ddH2O
3.3 ul 10x Expand Buffer "2"
3.3 ul dNTPs (2mM in each)
1 ul Oligo 1, 10 uM
1 ul Oligo 2, 10uM
0.5 ul, Expand polymerase "1"
0.5 ul Template ENA
march 6: Run gel on pcr products that require soeing (leucine and alanaine)
1. run gel on leu1, leu2, ala1, ala2.
2. make second gel, run remaining 9 samples for diagnostics.
3. gel purify leucine and alanine, zymoclean other pcr products
4. run two pcrs for soeing
User:Cheryl He 15:05, 18 March 2014 (EDT)
Last couple of days:
Completed soeing pcrs successfully
Digest of all pcr products (did not digest enough vector)
run gel of digested product, melt in ADB
Cheryl He 13:20, 20 March 2014 (EDT)
Run gel to double clarify contents of two tubes (vector or asp) Zymocleanup of vector