Team:Osaka/Notebook
From 2010.igem.org
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#* ''we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded'' | #* ''we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded'' | ||
# 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector | # 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector | ||
- | #* ligation product designated as | + | #* ligation product designated as 001; Chloramphenicol resistance |
#* same ligation mix composition as yesterday's | #* same ligation mix composition as yesterday's | ||
- | # Transformation of | + | # Transformation of 001 with pre-incubation for 1.5hr instead of 1hr |
===August 22 (Sun)=== | ===August 22 (Sun)=== | ||
- | # Transfer of | + | # Transfer of 001 to culture solution; incubation at 30°C (''why??'') |
# Transformation of the following parts: | # Transformation of the following parts: | ||
{| | {| | ||
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===August 23 (Mon)=== | ===August 23 (Mon)=== | ||
# Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture | # Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture | ||
- | # Miniprep & 'Cut check' of | + | # Miniprep & 'Cut check' of 001 |
#* cut at EcoRI, SpeI | #* cut at EcoRI, SpeI | ||
#* gel run with DNA ladder, digested plasmid, undigested plasmid | #* gel run with DNA ladder, digested plasmid, undigested plasmid | ||
#* (RESULTS?) | #* (RESULTS?) | ||
- | # Transfer of 3 more colonies of | + | # Transfer of 3 more colonies of 001 to liquid solution (''to store as glycerol stock - see Tue notes'') |
# Transformation of the following registry parts | # Transformation of the following registry parts | ||
{| | {| | ||
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#* (RESULTS) | #* (RESULTS) | ||
# Transfer of F1 to solution culture (''why?'') | # Transfer of F1 to solution culture (''why?'') | ||
- | # Preparation of glycerol stock of cell culture containing | + | # Preparation of glycerol stock of cell culture containing 001 (''why?'') |
#* 200ml of culture solution mixed with 100ml of 50% glycerol | #* 200ml of culture solution mixed with 100ml of 50% glycerol | ||
#* stored at -80°C | #* stored at -80°C | ||
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#* Inserts seem to be present in all samples | #* Inserts seem to be present in all samples | ||
# 3A assembly ligations: | # 3A assembly ligations: | ||
- | ## 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as | + | ## 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as 002 |
- | ## 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as | + | ## 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as 003 |
#* 2-2O using XbaI, PstI digest from yesterday | #* 2-2O using XbaI, PstI digest from yesterday | ||
#* 1-5A has Kan resistance | #* 1-5A has Kan resistance | ||
#* ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80°C | #* ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80°C | ||
- | # Transformation of ligation products | + | # Transformation of ligation products 002 and 003 |
===August 30 (Mon)=== | ===August 30 (Mon)=== | ||
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# Gel electrophoresis of the digests to confirm inserts | # Gel electrophoresis of the digests to confirm inserts | ||
#* all OK | #* all OK | ||
- | # Transfer of | + | # Transfer of 002 and 003 to solution culture (3 colonies each) |
===August 31 (Tue)=== | ===August 31 (Tue)=== | ||
- | # Miniprep of | + | # Miniprep of 002, 003 |
- | # Cut check of | + | # Cut check of 002, 003 with EcoRI, SpeI |
- | #* | + | #* 003 was properly cut, but the insert length was inconsistent; looking back at 8/28 gel result, length of 2-2O (downstream part in 003) also seemed to be longer than expected |
# Repeat colony pick-up and solution culture of 2-2O (5 colonies this time) | # Repeat colony pick-up and solution culture of 2-2O (5 colonies this time) | ||
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#* 1-12B did not transform successfully | #* 1-12B did not transform successfully | ||
# 3A assembly ligations: | # 3A assembly ligations: | ||
- | ## | + | ## 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004 |
- | ## | + | ## 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005 |
# Transformation of ligation products | # Transformation of ligation products | ||
===September 3 (Fri)=== | ===September 3 (Fri)=== | ||
- | # Colony check: yesterday's transformations seem to have failed; repeat of transformations of | + | # Colony check: yesterday's transformations seem to have failed; repeat of transformations of 004 and 005 with 50μl competent cells, 2μl ligation product (''note: colonies appeared later; these repeats were then discarded'') |
# Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI | # Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI | ||
#* all lengths ok | #* all lengths ok | ||
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===September 4 (Sat)=== | ===September 4 (Sat)=== | ||
- | # Miniprep of | + | # Miniprep of 004, 005 |
- | # Restriction digest of | + | # Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI |
# Gel electrophoresis | # Gel electrophoresis | ||
#* 1-7D -> OK | #* 1-7D -> OK | ||
- | #* | + | #* 004 -> insert length same as 001; ''since both upstream part 001 and vector 1-3A were C resistance, 3A assembly must have failed to yield ligation product; try Standard Assembly!'' |
- | #* | + | #* 005 -> ?? |
- | # Gel electrophoresis followed by purification of | + | # Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly |
#* gel purification performed according to protocol in QIAquick Spin Handbook | #* gel purification performed according to protocol in QIAquick Spin Handbook | ||
- | # Ligation of gel-purified | + | # Ligation of gel-purified 001 to 1-2J or 2-22P, with vector 1-3A, to make 004 or 005 respectively (''same 004 and 005 as designed before'') |
===September 5 (Sun)=== | ===September 5 (Sun)=== | ||
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===September 7 (Tue)=== | ===September 7 (Tue)=== | ||
- | # Miniprep of | + | # Miniprep of 004, 005 |
# Cut check with EcoRI, SpeI | # Cut check with EcoRI, SpeI | ||
#* both insert lengths ok! | #* both insert lengths ok! | ||
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|} | |} | ||
# Transfer to solution culture | # Transfer to solution culture | ||
- | #* | + | #* 004, 005 transformed on 9/5 (pick up from fresh colonies) -> ''needed more plasmid'' |
#* 2-10F, 2-10H transformed yesterday | #* 2-10F, 2-10H transformed yesterday | ||
+ | |||
+ | ===September 8 (Wed)=== | ||
+ | # Miniprep 2-10F, 2-10H, 004, 005 | ||
+ | |||
+ | 2.制限酵素処理 | ||
+ | 2-10F,2-10H,004,005 | ||
+ | ネガティブコントロール | ||
+ | プラスミド 2.5μl 2.5μl | ||
+ | EcoRI 0.5μl 0.5μl | ||
+ | Spe I 0.5μl 0μl | ||
+ | 10×NEbuffer2 5μl 5μl | ||
+ | 100×BSA 0.5μl 0.5μl | ||
+ | H2O 41μl 41.5μl | ||
+ | total 50μl 50μl | ||
+ | |||
+ | 37℃ incubate 30min | ||
+ | |||
+ | 3.電気泳動 | ||
+ | dye 2μl、サンプル 10μl、Ladder 2μl | ||
+ | 1%Agalose gel、100V 20min, EtBr 1h10min | ||
+ | EtBrは新しいものにかえた。10μg/μl 30μlを300mlに | ||
+ | |||
+ | 005 005 004 004 2-10H 2-10H 2-10F 2-10F Ladder | ||
+ | E ES E ES E ES E ES | ||
+ | |||
+ | 4.PCR | ||
+ | mix組成 | ||
+ | Ex taq 0.25μl | ||
+ | 10×taq Buffer 5μl | ||
+ | dNTP Mix 4μl | ||
+ | Template 1μl | ||
+ | primer rev 0.5μl | ||
+ | fwr 0.5μl | ||
+ | DSW 38.75μl | ||
+ | total 50μl | ||
+ | |||
+ | 94℃ 2min | ||
+ | 30サイクル | ||
+ | 94℃ 30sec tube6 cenA | ||
+ | 68℃ 30sec tube7 cex | ||
+ | 72℃ 2min tube8 bgl | ||
+ | |||
+ | 5.電気泳動 | ||
+ | dye 2μl、サンプル 5μl、Ladder 2μl | ||
+ | 1%Agalose gel、100V , EtBr | ||
+ | Ladder, 6 ,7 ,8 PCR後そのまま | ||
+ | |||
+ | 6.電気泳動 | ||
+ | dye 2μl、サンプル 10μl、Ladder 2μl | ||
+ | 1%Agalose gel、100V (PCR purification Ladder) | ||
+ | 左から8(プラスミド)7(プラスミド)6(プラスミド) 8(PCR)7(PCR)6(PCR)Ladder | ||
+ | |||
+ | PCRができていない。 Templateのみがバンドで見られる。 | ||
+ | 8の8kbpのバンドは何? | ||
+ | |||
+ | 7.再度PCR | ||
+ | Ex taq 0.25μl | ||
+ | 10×taq Buffer 5μl | ||
+ | dNTP Mix 4μl | ||
+ | Template 1μl | ||
+ | primer rev 0.5μl | ||
+ | fwr 0.5μl | ||
+ | DSW 38.75μl | ||
+ | total 50μl | ||
+ | |||
+ | tube 1: F1×100 | ||
+ | 2: F1×1000 | ||
+ | 3: 2-20H×100 | ||
+ | 4: 2-20H×1000 | ||
+ | 5: 2-20J×100 | ||
+ | 6: 2-20J×1000 | ||
+ | |||
+ | 8. A01,A02,A03 を液培へ | ||
+ | 左2つにAmp 3μl 残りにK 0.6μl | ||
+ | →37℃ incubate 23:10~ | ||
+ | |||
+ | 9.電気泳動 | ||
+ | 5×dye 1μl、サンプル 5μl、 マーカー2μl | ||
+ | 1%Agalose gel、100V 25min, EtBr 20min | ||
+ | |||
+ | PCR産物が見られない。アニーリング温度が原因か? | ||
+ | |||
+ | 10.再再度PCR | ||
+ | mix組成 | ||
+ | Ex taq 0.25μl | ||
+ | 10×taq Buffer 5μl | ||
+ | dNTP Mix 4μl | ||
+ | Template 1μl | ||
+ | primer rev 0.5μl | ||
+ | fwr 0.5μl | ||
+ | DSW 38.75μl | ||
+ | total 50μl | ||
+ | |||
+ | サンプル番号 | ||
+ | 1. F1×10 94℃ 1min | ||
+ | 2. 2-20H×10 → 98℃ 10sec | ||
+ | 3. 2-20J×10 65℃ 30sec 下3つを30cycle | ||
+ | 72℃ 2min | ||
+ | |||
+ | 4. F1×10 94℃ 2min | ||
+ | 5.2-20H×10 → 98℃ 10sec 30cycle | ||
+ | 6.2-20J×10 68℃ 2min | ||
+ | 4.プライマー異なるものも入ってしまった テンプレート入れすぎ | ||
+ | |||
+ | 11.再々度PCR 電気泳動 | ||
+ | 以下9と同じ | ||
+ | 左からLadder Sample 1 2 3 4 5 6 | ||
+ | |||
+ | Annealing temp 低いか? | ||
+ | ※ 今後PCRの条件をどう設定していくか? | ||
+ | |||
+ | ゲルの結果 | ||
+ | Sample1:16721bp(これは正解) | ||
+ | Sample2:これは失敗 | ||
+ | Sample3:1350bp付近にバンドあり。目的遺伝子は増幅できた。 | ||
+ | Sample4: | ||
+ | Sample5:1461bp付近にバンドあり。目的遺伝子は増幅できた。 | ||
+ | Sample6:1353bp付近にバンドあり。目的遺伝子は増幅できた。 | ||
+ | |||
+ | Sample2を除き 45μl電気泳動 Gel purification を行うことにした。 | ||
+ | この時点でF1はprimerの設計ミスにより、Assemblyの対象から | ||
+ | 外し、破棄した。(Sample1,Sample4 破棄) | ||
+ | |||
+ | 12.Gel purification | ||
+ | 再々度PCR Sample 3, 5 ,6 ,8を 電気泳動 Gel purification | ||
+ | ladder 4μl | ||
+ | loading 41 | ||
+ | |||
Revision as of 13:40, 8 October 2010
Calendar
July | ||||||
S | M | T | W | T | F | S |
1 | 2 | 3 | ||||
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
August | ||||||
S | M | T | W | T | F | S |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 | ||||
September | ||||||
S | M | T | W | T | F | S |
1 | 2 | 3 | 4 | |||
5 | 6 | 7 | 8 | 9 | 10 | 11 |
12 | 13 | 14 | 15 | 16 | 17 | 18 |
19 | 20 | 21 | 22 | 23 | 24 | 25 |
26 | 27 | 28 | 29 | 30 | ||
October | ||||||
S | M | T | W | T | F | S |
1 | 2 | |||||
3 | 4 | 5 | 6 | 7 | 8 | 9 |
10 | 11 | 12 | 13 | 14 | 15 | 16 |
17 | 18 | 19 | 20 | 21 | 22 | 23 |
24 | 25 | 26 | 27 | 28 | 29 | 30 |
31 | ||||||
November | ||||||
S | M | T | W | T | F | S |
1 | 2 | 3 | 4 | 5 | 6 | |
7 | 8 | 9 | 10 | 11 | 12 | 13 |
14 | 15 | 16 | 17 | 18 | 19 | 20 |
21 | 22 | 23 | 24 | 25 | 26 | 27 |
28 | 29 | 30 | ||||
Notebook
July 29 (Thu)
Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura
- Safety lecture for junior members.
- Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).
July 31 (Sat)
Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka
- Meeting
- Summer project schedule
- List of genes to clone
August 2 (Mon)
Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka
- Culture medium preparation
- LB agar plates (49 antibiotic-less plates)
- LB liquid medium (500 ml)
- Competent cells preparation - Nojima Method
- SOB medium (MgCl2 not yet added) -> stored at 4˚C
- TB buffer -> stored at 4˚C
- Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n
Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.
August 3 (Tue)
Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh
- Competent cells preparation (continued)
- Preparation of glucose solution for making SOC medium.
- Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
- (Night) Transfer from pre-culture to growth culture.
August 4 (Wed)
Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh
- OD measurements throughout the day till required OD (0.3~0.7) was obtained.
- Completion of competent cells according to protocol.
August 5 (Thu)
Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino
- Transformation of Registry parts:
ID | Part Name | Resistance | Description |
---|---|---|---|
2-20J | <bbpart>BBa_K118023</bbpart> | A | C. fermi endocellulase Cen A coding |
2-20H | <bbpart>BBa_K118022</bbpart> | A | C. fermi exocellulase Cex coding |
1-2M | <bbpart>BBa_B0034</bbpart> | A | RBS |
1-13D | <bbpart>BBa_B0010</bbpart> | A | terminator |
1-1D | <bbpart>BBa_R0010</bbpart> | A | promoter |
1-18F | <bbpart>BBa_E1010</bbpart> | K | RFP coding |
Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.
August 6 (Fri)
Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh
- Colony check
- All transformed cells produced colonies!
- Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
- Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C
August 7 (Sat)
Attendance: Nakamura, Saka, Yasumoto, Takino
- Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
- Transformation of construction plasmids
ID | Part Name | Resistance | Description |
---|---|---|---|
1-1C | <bbpart>pSB1A3</bbpart> | A | construction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>) |
1-3A | <bbpart>pSB1C3</bbpart> | C | (" ") |
1-5A | <bbpart>pSB1K3</bbpart> | K | (" ") |
- Meeting
August 8 (Sun)
Attendance: Nakamura, Yasumoto
- Colony check
- All parts successfully transformed
- Transfer to LB culture medium
August 9 (Mon)
- Miniprep of 1-1C
- Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
- (WHICH ENZYMES?)
- Gel electrophoresis of digests ("cut check")
- 2-20H, 2-20J, 1-1C -> OK
- 1-1D, 1-18F, 1-13D, 1-2M -> not cut (single band, MW approx. equal to vector + insert)
- Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
- Yesterday's inoculated culture mediums contained the wrong antibiotics!
- Transformation of <bbpart>BBa_I13521</bbpart>, <bbpart>BBa_I13522</bbpart>, <bbpart>BBa_I13600</bbpart>, <bbpart>BBa_K204031</bbpart>, <bbpart>BBa_K204051</bbpart>, <bbpart>BBa_K204032</bbpart> from last year's stock
August 10 (Tue)
- Miniprep of 1-3A, 1-5A
- Restriction digests of 1-3A, 1-5A
- Gel electrophoresis
- 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
- 1-3A, 1-5A -> OK; others -> not cut (again)
- 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
- all parts not cut
- 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
August 11 (Wed)
- Miniprep of last year's parts transformed on Monday
- Gel electrophoresis of 1-1D, 1-18F, 1-13D, 1-2M (3rd repeat!!!)
- all 4 not cut... AGAIN
- so far all restriction digests involving XbaI seem to have failed; problem with enzyme stock?
- will try with different set of restriction enzymes next week
- Sent miniprepped last year's parts to ECUST team in Shanghai, China
August 16 (Mon)
- Restriction digests
- 1-13D, 1-2M, 1-1D, 1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
- 2-20J with XbaI, PstI to check/confirm XbaI activity
- Gel electrophoresis of digests
- (RESULTS?)
- Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D, 1-2M, 1-1D, 1-18F
- 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added
August 17 (Tue)
- Mini-prep of 1-13D, 1-2M, 1-1D, 1-18F parts inoculated yesterday (2 of each)
- 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
- 1-1D, 1-18F with EcoRI, SpeI
- 1-13D, 1-2M with XbaI, PstI
- (RESULTS?)
- Transformation of secretion tag parts using 25μl of competent cells each
ID | Part Name | Resistance | Description |
---|---|---|---|
2-22P | <bbpart>BBa_K103006</bbpart> | A | OmpA outer membrane protein + linker |
1-2J | <bbpart>BBa_J32015</bbpart> | A,K | PelB leader sequence |
August 18 (Wed)
iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda
August 19 (Thu)
- Transfer of 2-22P, 1-2J to solution culture
- Gel electrophoresis of digests from 'cut check' products from Tuesday
- repeat run, but each digest together with undigested plasmid DNA)
- 2% agarose gel instead of the usual 1%
- (RESULTS?)
- Gel electrophoresis of 1-1D digest only
- (RESULT?)
- Multiple restriction digests of 1-1D to check for problems at restriction sites
- tried the following: EcoRI only; SpeI only; EcoRI + SpeI
- Night: miniprep of 2-22P, 1-2J inoculated in the morning
August 20 (Fri)
- Gel electrophoresis of 1-1D and its digests
- (RESULTS?)
- 'Cut check' of parts miniprepped the night before
- both 2-22P & 1-2J cut with XbaI, PstI
- enzyme inactivation at 80°C, 20min
- (RESULTS?)
- Restriction digest of 2-20J (WHICH ENZYMES?)
- Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
- reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
- reaction at room temperature for 10min; ligase inactivation at 80°C for 20min
- Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix
August 21 (Sat)
- Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
- we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
- 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
- ligation product designated as 001; Chloramphenicol resistance
- same ligation mix composition as yesterday's
- Transformation of 001 with pre-incubation for 1.5hr instead of 1hr
August 22 (Sun)
- Transfer of 001 to culture solution; incubation at 30°C (why??)
- Transformation of the following parts:
ID | Part Name | Resistance | Description |
---|---|---|---|
2-4A | <bbpart>BBa_J63005</bbpart> | A | yeast ADH1 promoter |
F1 | N/A | A | beta-galactosidase from Edinburgh team |
F2 | N/A | C | RBS + F1 |
F3 | N/A | C | Lac promoter + RBS + F1 |
- O/N incubation at 37°C as per normal
August 23 (Mon)
- Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
- Miniprep & 'Cut check' of 001
- cut at EcoRI, SpeI
- gel run with DNA ladder, digested plasmid, undigested plasmid
- (RESULTS?)
- Transfer of 3 more colonies of 001 to liquid solution (to store as glycerol stock - see Tue notes)
- Transformation of the following registry parts
ID | Part Name | Resistance | Description |
---|---|---|---|
2-2O | <bbpart>J63003</bbpart> | A | yeast Kozak sequence |
3-11I | <bbpart>K105027</bbpart> | A | 'cyc100' minimal promoter |
August 24 (Tue)
- Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
- transfer to solution culture
- Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
- (RESULTS)
- Transfer of F1 to solution culture (why?)
- Preparation of glycerol stock of cell culture containing 001 (why?)
- 200ml of culture solution mixed with 100ml of 50% glycerol
- stored at -80°C
August 25 (Wed)
- Miniprep of parts in solution culture: 2-2O, 3-11I, F1
- Cut check of 3-11I & F1 with EcoRI, SpeI
- (RESULTS?)
August 26 (Thu)
- Transformation of <bbpart>BBa_K204022</bbpart>, <bbpart>BBa_K204025</bbpart>, and <bbpart>BBa_K204040</bbpart> to send to Shanghai ECUST team in China.
August 27 (Fri)
- Transfer of yesterday's transformed parts (all produced colonies) to solution culture
- Transformation of the following parts
ID | Part Name | Resistance | Description |
---|---|---|---|
1-1K | <bbpart>BBa_J63010</bbpart> | A | Protein fusion vector (Silver standard) |
1-1I | <bbpart>BBa_J63009</bbpart> | A | Low copy protein fusion vector (Silver standard) |
3-3G | <bbpart>BBa_K157013</bbpart> | A | 15aa glycine-serine linker (Freiburg standard) |
- using competent cells opened on 8/20
- Preparation of YPD yeast culture medium with the following recipe:
MiliQ water | 1 liter | |
Bacto tryptone | 20.0g | 2% |
Bacto yeast extract | 10.0g | 1% |
Glucose | 20.0g | 2% |
- pH was adjusted to 5.8
- autoclaved before use
- 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
- Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar
August 28 (Sat)
- Miniprep of parts in solution culture
- Restriction digest (for cut check) - 37°C for 30min
- 2-4A & 3-11I with EcoRI, SpeI
- 2-2O with XbaI, PstI
- K204022, K204025, K204040 wih EcoRI, PstI
- Gel electrophoresis of digests
- Plasmids not detected for 2-4A & 3-11I - mistake during miniprep? culture duration too long, plasmid loss occurred?
- Transfer of the following parts to solution culture
- 1-1K, 1-1I, 3-3G (yesterday's transformations)
- 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)
August 29 (Sun)
- Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
- Restriction digests
- for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
- for assembly: 2-4A, 3-11I with EcoRI, SpeI (upstream parts)
- Gel electrophoresis for confirmation
- Inserts seem to be present in all samples
- 3A assembly ligations:
- 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as 002
- 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as 003
- 2-2O using XbaI, PstI digest from yesterday
- 1-5A has Kan resistance
- ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80°C
- Transformation of ligation products 002 and 003
August 30 (Mon)
- Restriction digests for 3A assembly
- 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
- 2-20J (CenA), 2-20H (Cex), F1 (BglX) with XbaI, PstI
- beta-glucosidase received from Edinburgh team temporarily designated BglX
- Gel electrophoresis of the digests to confirm inserts
- all OK
- Transfer of 002 and 003 to solution culture (3 colonies each)
August 31 (Tue)
- Miniprep of 002, 003
- Cut check of 002, 003 with EcoRI, SpeI
- 003 was properly cut, but the insert length was inconsistent; looking back at 8/28 gel result, length of 2-2O (downstream part in 003) also seemed to be longer than expected
- Repeat colony pick-up and solution culture of 2-2O (5 colonies this time)
September 1 (Wed)
- Transformation
ID | Part Name | Resistance | Description |
---|---|---|---|
1-12D | <bbpart>BBa_E2030</bbpart> | K | yeast-optimized EYFP |
1-12B | <bbpart>BBa_E2020</bbpart> | K | yeast-optimized ECFP |
3-2K | <bbpart>BBa_K165001</bbpart> | A | yeast GAL1 promoter |
1-7D | <bbpart>BBa_J63006</bbpart> | A | yeast GAL1 promoter + Kozak sequence |
- Miniprep of 5 separate cultures of 2-2O inoculated yesterday
- Cut check of 2-2O with XbaI, PstI
- 0.7kbp bands in all 5 samples even though insert is supposed to be only 18bp - problem with the part (inconsistency confirmed from registry info page)
- obtain Kozak sequence by PCR instead?
September 2 (Thu)
- Colony check
- 1-12D, 3-2K, 1-7D produced colonies -> inoculated into solution culture
- 1-12B did not transform successfully
- 3A assembly ligations:
- 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004
- 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005
- Transformation of ligation products
September 3 (Fri)
- Colony check: yesterday's transformations seem to have failed; repeat of transformations of 004 and 005 with 50μl competent cells, 2μl ligation product (note: colonies appeared later; these repeats were then discarded)
- Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI
- all lengths ok
- Transfer of yesterday's transformations (colonies appeared later in the evening) to culture solution (2 colonies picked up from each plate)
September 4 (Sat)
- Miniprep of 004, 005
- Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI
- Gel electrophoresis
- 1-7D -> OK
- 004 -> insert length same as 001; since both upstream part 001 and vector 1-3A were C resistance, 3A assembly must have failed to yield ligation product; try Standard Assembly!
- 005 -> ??
- Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly
- gel purification performed according to protocol in QIAquick Spin Handbook
- Ligation of gel-purified 001 to 1-2J or 2-22P, with vector 1-3A, to make 004 or 005 respectively (same 004 and 005 as designed before)
September 5 (Sun)
- Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.
September 6 (Mon)
- Transfer of yesterday's transformations to solution culture
- Transformation of the following registry parts:
ID | Part Name | Resistance | Description |
---|---|---|---|
2-10F | <bbpart>BBa_K081005</bbpart> | A | constitutive promoter + RBS |
2-10H | <bbpart>BBa_K081006</bbpart> | A | lambda phage promoter + RBS |
September 7 (Tue)
- Miniprep of 004, 005
- Cut check with EcoRI, SpeI
- both insert lengths ok!
- Transformation of DNA for PGA synthesis-related genes
ID | Part Name | Resistance | Description |
---|---|---|---|
A01 | pTPG01-1 | A | plasmid pTrc99A with pgs genes inserted |
A02 | pTPG01-2 | A | '' |
A03 | pBSGR3 | K | glutamine racemase |
- Transfer to solution culture
- 004, 005 transformed on 9/5 (pick up from fresh colonies) -> needed more plasmid
- 2-10F, 2-10H transformed yesterday
September 8 (Wed)
- Miniprep 2-10F, 2-10H, 004, 005
2.制限酵素処理
2-10F,2-10H,004,005
ネガティブコントロール プラスミド 2.5μl 2.5μl EcoRI 0.5μl 0.5μl Spe I 0.5μl 0μl 10×NEbuffer2 5μl 5μl 100×BSA 0.5μl 0.5μl H2O 41μl 41.5μl total 50μl 50μl
37℃ incubate 30min
3.電気泳動 dye 2μl、サンプル 10μl、Ladder 2μl
1%Agalose gel、100V 20min, EtBr 1h10min EtBrは新しいものにかえた。10μg/μl 30μlを300mlに
005 005 004 004 2-10H 2-10H 2-10F 2-10F Ladder
E ES E ES E ES E ES
4.PCR mix組成
Ex taq 0.25μl 10×taq Buffer 5μl dNTP Mix 4μl Template 1μl primer rev 0.5μl fwr 0.5μl DSW 38.75μl total 50μl
94℃ 2min
30サイクル
94℃ 30sec tube6 cenA 68℃ 30sec tube7 cex 72℃ 2min tube8 bgl
5.電気泳動
dye 2μl、サンプル 5μl、Ladder 2μl 1%Agalose gel、100V , EtBr Ladder, 6 ,7 ,8 PCR後そのまま
6.電気泳動 dye 2μl、サンプル 10μl、Ladder 2μl
1%Agalose gel、100V (PCR purification Ladder)
左から8(プラスミド)7(プラスミド)6(プラスミド) 8(PCR)7(PCR)6(PCR)Ladder
PCRができていない。 Templateのみがバンドで見られる。
8の8kbpのバンドは何?
7.再度PCR
Ex taq 0.25μl 10×taq Buffer 5μl dNTP Mix 4μl Template 1μl primer rev 0.5μl fwr 0.5μl DSW 38.75μl total 50μl
tube 1: F1×100 2: F1×1000 3: 2-20H×100 4: 2-20H×1000 5: 2-20J×100 6: 2-20J×1000
8. A01,A02,A03 を液培へ
左2つにAmp 3μl 残りにK 0.6μl
→37℃ incubate 23:10~
9.電気泳動
5×dye 1μl、サンプル 5μl、 マーカー2μl 1%Agalose gel、100V 25min, EtBr 20min
PCR産物が見られない。アニーリング温度が原因か?
10.再再度PCR
mix組成 Ex taq 0.25μl 10×taq Buffer 5μl dNTP Mix 4μl Template 1μl primer rev 0.5μl fwr 0.5μl DSW 38.75μl total 50μl
サンプル番号
1. F1×10 94℃ 1min 2. 2-20H×10 → 98℃ 10sec 3. 2-20J×10 65℃ 30sec 下3つを30cycle 72℃ 2min
4. F1×10 94℃ 2min 5.2-20H×10 → 98℃ 10sec 30cycle 6.2-20J×10 68℃ 2min
4.プライマー異なるものも入ってしまった テンプレート入れすぎ
11.再々度PCR 電気泳動 以下9と同じ 左からLadder Sample 1 2 3 4 5 6
Annealing temp 低いか? ※ 今後PCRの条件をどう設定していくか?
ゲルの結果
Sample1:16721bp(これは正解) Sample2:これは失敗 Sample3:1350bp付近にバンドあり。目的遺伝子は増幅できた。 Sample4: Sample5:1461bp付近にバンドあり。目的遺伝子は増幅できた。 Sample6:1353bp付近にバンドあり。目的遺伝子は増幅できた。
Sample2を除き 45μl電気泳動 Gel purification を行うことにした。 この時点でF1はprimerの設計ミスにより、Assemblyの対象から
外し、破棄した。(Sample1,Sample4 破棄)
12.Gel purification 再々度PCR Sample 3, 5 ,6 ,8を 電気泳動 Gel purification
ladder 4μl loading 41