Sbb14-nsc

25Feb2014

Oligos with restriction sites

Name Oligo (incorporate NcoI site into the start codon as discussed earlier) Thermus-Ala-Forward-- gtcggtCGTCTCccatgGCGcgcacggcggagatccgcga

Thermus-Ala-Reverse gtcggtCGTCTCgaattcctaggggaggaggccggggag

Thermus-Cys-Forward gtcggtGGTCTCccatgggcctggtcatctacgac

Thermus-Cys-Reverse gtcggtGGTCTCgaattcttagcgctccaggcgccacc

Thermus-Asp-Forward gtcggtGGTCTCccatgGCGcgtcgcacccactacgccgg

Thermus-Asp-Reverse gtcggtGGTCTCgaattctcatggccggaccaccatgag

Thermus-Glu-Forward gtcggtCCATGGtggtgacccgcatcgc

Thermus-Glu-Reverse gtcggtGAATTCctaggcgagggcccgctcca

Thermus-Phe-Forward gtcggtGGTCTCccatggtggaagaagccttggccgcc

Thermus-Phe-Reverse gtcggtGGTCTCgaattctcatagaacccccttgaactgc

Thermus-Gly-Forward gtcggtGGTCTCccatgGCGcctgcgagcagcctggacgaac

Thermus-Gly-Reverse gtcggtGGTCTCgaattcctaccacctaagcctctcccg

Thermus-His-Forward gtcggtGGTCTCccatgGCGaccgctcgggccgtgcgggg

Thermus-His-Reverse gtcggtGGTCTCgaattctcacccgagggcctggagga

Thermus-Ile-Forward gtcggtccatgGCGttcaaggaggtcggcgagcc

Thermus-Ile-Reverse gtcggtGAATTCtcactccgcccgggccaggt

Thermus-Lys-Forward gtcggtccatgGCGaacgaccagacccgccagcg

Thermus-Lys-Reverse gtcggtGAATTCttagaccccttcttccaccgc

Thermus-Leu-Forward gtcggtCGTCTCccatggagaagtacaacccgcacg

Thermus-Leu-Reverse gtcggtCGTCTCgaattcctacccccgcactaccaggt

10Mar2014 Gel actual lengths Ala A 2649 Cys C 1443 Asp D 1743 Glu E 1407 Phe F 1053 Gly G 1521 His H 1266 Ile I 3132 Lys K 1479 Leu L 2637 Actual Gels

25Feb2014 Oligos with restriction sites

Name Oligo (incorporate NcoI site into the start codon as discussed earlier)

Thermus-Ala-Forward gtcggtCGTCTCccatgGCGcgcacggcggagatccgcga

Thermus-Ala-Reverse gtcggtCGTCTCgaattcctaggggaggaggccggggag

Thermus-Cys-Forward gtcggtGGTCTCccatgggcctggtcatctacgac

Thermus-Cys-Reverse gtcggtGGTCTCgaattcttagcgctccaggcgccacc

Thermus-Asp-Forward gtcggtGGTCTCccatgGCGcgtcgcacccactacgccgg

Thermus-Asp-Reverse gtcggtGGTCTCgaattctcatggccggaccaccatgag

Thermus-Glu-Forward gtcggtCCATGGtggtgacccgcatcgc

Thermus-Glu-Reverse gtcggtGAATTCctaggcgagggcccgctcca

Thermus-Phe-Forward gtcggtGGTCTCccatggtggaagaagccttggccgcc

Thermus-Phe-Reverse gtcggtGGTCTCgaattctcatagaacccccttgaactgc

Thermus-Gly-Forward gtcggtGGTCTCccatgGCGcctgcgagcagcctggacgaac

Thermus-Gly-Reverse gtcggtGGTCTCgaattcctaccacctaagcctctcccg

Thermus-His-Forward gtcggtGGTCTCccatgGCGaccgctcgggccgtgcgggg

Thermus-His-Reverse gtcggtGGTCTCgaattctcacccgagggcctggagga

Thermus-Ile-Forward gtcggtccatgGCGttcaaggaggtcggcgagcc

Thermus-Ile-Reverse gtcggtGAATTCtcactccgcccgggccaggt

Thermus-Lys-Forward gtcggtccatgGCGaacgaccagacccgccagcg

Thermus-Lys-Reverse gtcggtGAATTCttagaccccttcttccaccgc

Thermus-Leu-Forward gtcggtCGTCTCccatggagaagtacaacccgcacg

Thermus-Leu-Reverse gtcggtCGTCTCgaattcctacccccgcactaccaggt

10Mar2014 Gel actual lengths Ala A 2649 Cys C 1443 Asp D 1743 Glu E 1407 Phe F 1053 Gly G 1521 His H 1266 Ile I 3132 Lys K 1479 Leu L 2637 Actual Gels

PCR in practice 11March2014 10x dilution Blue top Added 100micromolar water Second tube Added 1:9 oligo to water Next Thursday 13March2014 PCR for A I L Did PCR for C D H Run gels For no showing gels use DMSO Oligos that are off 11march A c d h I l Alanine-too short Aspartate-either ran over or there is nothing in there Cysteine Histadine-too short Isoleucine-very faint but readable so could have screwed up but is correct #kb What we did for our reactions.

11March2014 Restarted, setup PCR 13March2014 Gel Ladder,a,c,d,h,I,l Zymo cleanup

18March2014 Maybe good oligos (iffy on the lengths) C, I, L Good Oligos E, F, G, K Bad Oligos H (too short), D (Not there at all), A (possibly two products)

Sequence (maybe good and good oligos)

C, I, L, F, G, K, E

Cut out band in A and purify Redilute A,C,D,H,I,L PCR A (DMSO) D (DMSO), H (DMSO), I(DSMO), L (with and without DMSO), C (without DMSO) Mastermix DMSO water-20.7 microLiter x6 = 124.2uL 3.3u: DMSO x6 = 19.8 3.3uL *6 expand buffer 2 = 19.8 3.3 micro dNTP x6 = 19.8 0.5 uL *6 template = 3uL Mastermix regular 24 micro water *2.5 = 60uL 3.3 micr dNTP *2.5 = 8.25 3.3uL buffer 2 *2.5 = 8.25uL 0.5uL template *2.5 = 1.25uL PCR programs Ran 4k55 L (DMSO), L, I(DMSO), A(DMSO) Run at 2k55 C, H (DMSO) Run at 2k45 D(DMSO)

20March2014 Planned: Analytical gel Load one lane with A from previous for doing a gel purification Some sort of zymo cleanup Done: redo PCR, because there were no Expand Polymerase

1April 2014 Gel x2 of PCR from 20 march Redo gels for a, c, d, h, i, l (with and without DMSO) One gel has oligos with enzyme added after the fact, and another with oligos with enzymes added as the pcr was being made Gel order for both: a c d h i l (no dmso) l

3april digest all oligos except for A and L (rerun gels) zymo cleanup Gel purify, then amplify gel sample of A

8April2014 digestion redesign A, L zymo

10april2014 PCR digest mini prep-then ligate with vector run gel

14Apr2014 PCR context amplification of A and I with and without DMSO

17Apr2014 redo digest Gel

18Apr2014 Gel Digestion

22Apr2014 Gel redo digests PCR amplification of A & I PCR A off of gel fragment Phusion reactions with two A zymoclean context amplification PCR products saving #3 that are supposed to be used for digestion and throwing away tubes which were digested

mastermix for all pcr (multiply by 7 for extra) PCR labelling 1 = gel 2 = context PCR 3 = used DMSO in PCR the # on the PCRs (volume of 50uL) ran at 4K55 Gel no good!

Gel on left is group 1, gel on right is group 2. The only potentially valid genes are in lanes 8 and 10 in group 1 and 3 and 4 in group 2.

24Apr2014 gel for analytical on PCR of A and I off of A and I context sequence and A gel purification gel for group 3 aa used SYBR instead of gel green

PCR in practice 11March2014 10x dilution Blue top Added 100micromolar water Second tube Added 1:9 oligo to water Next Thursday 13March2014 PCR for A I L Did PCR for C D H Run gels For no showing gels use DMSO Oligos that are off 11march A c d h I l Alanine-too short Aspartate-either ran over or there is nothing in there Cysteine Histadine-too short Isoleucine-very faint but readable so could have screwed up but is correct #kb What we did for our reactions.

11March2014 Restarted, setup PCR

13March2014 Gel Ladder,a,c,d,h,I,l Zymo cleanup

18March2014 Maybe good oligos (iffy on the lengths) C, I, L Good Oligos E, F, G, K Bad Oligos H (too short), D (Not there at all), A (possibly two products)

Sequence (maybe good and good oligos)

C, I, L, F, G, K, E

Cut out band in A and purify Redilute A,C,D,H,I,L PCR A (DMSO) D (DMSO), H (DMSO), I(DSMO), L (with and without DMSO), C (without DMSO) Mastermix DMSO water-20.7 microLiter x6 = 124.2uL 3.3u: DMSO x6 = 19.8 3.3uL *6 expand buffer 2 = 19.8 3.3 micro dNTP x6 = 19.8 0.5 uL *6 template = 3uL Mastermix regular 24 micro water *2.5 = 60uL 3.3 micr dNTP *2.5 = 8.25 3.3uL buffer 2 *2.5 = 8.25uL 0.5uL template *2.5 = 1.25uL PCR programs Ran 4k55 L (DMSO), L, I(DMSO), A(DMSO) Run at 2k55 C, H (DMSO) Run at 2k45 D(DMSO)

20March2014 Planned: Analytical gel Load one lane with A from previous for doing a gel purification Some sort of zymo cleanup Done: redo PCR, because there were no Expand Polymerase

1April 2014 Gel x2 of PCR from 20 march Redo gels for a, c, d, h, i, l (with and without DMSO) One gel has oligos with enzyme added after the fact, and another with oligos with enzymes added as the pcr was being made Gel order for both: a c d h i l (no dmso) l

3april digest all oligos except for A and L (rerun gels) zymo cleanup Gel purify, then amplify gel sample of A

8April2014 digestion redesign A, L zymo

10april2014 PCR digest mini prep-then ligate with vector run gel

14Apr2014 PCR context amplification of A and I with and without DMSO

17Apr2014 redo digest Gel

18Apr2014 Gel Digestion

22Apr2014 Gel redo digests PCR amplification of A & I PCR A off of gel fragment Phusion reactions with two A zymoclean context amplification PCR products saving #3 that are supposed to be used for digestion and throwing away tubes which were digested

mastermix for all pcr (multiply by 7 for extra) PCR labelling 1 = gel 2 = context PCR 3 = used DMSO in PCR the # on the PCRs (volume of 50uL) ran at 4K55 Gel no good!

Gel on left is group 1, gel on right is group 2. The only potentially valid genes are in lanes 8 and 10 in group 1 and 3 and 4 in group 2.

24Apr2014 gel for analytical on PCR of A and I off of A and I context sequence and A gel purification gel for group 3 aa used SYBR instead of gel green

Gel order C,D,E,F,G,H,I,K,L