Team:Freiburg/Notebook/standardisation

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Assembly of Biobricks Notebook

Theoretical cloning

Cas9 primers were design to exchange several bases - all as silent mutations – in order to remove all restriction recognition sides of the iGEM enzymes EcoI, NotI, XbaI, NgoMIV, SpeI, PstI, AgeI. Then the standardized Cas9 was brought into pSB1C3 (RFC25) and was used for fusion with with other parts.

Practical work

June

17.06.2013

PCRs for Gibson assembly 1:

PCR1: oIG22+oIG28 PCR2: oIG29+oIG26 PCR3: oIG27+oIG24 PCR4: oIG25+oIG23/

Template: pX334 Annealing temperature dependent of fragment size:

PCR1: 1130 bp
PCR2: 1369 bp
PCR3: 314 bp
PCR4: 1409 bp

PCR mixture

per tube (50 µl)
10 µl	Q5 reaction buffer
10 µl	GC enhancer
4 µl	dNTPs
0.5 µl	Q5 Polymerase
22.5 µl	add with water up to 48 µl

per reaction add 1 µl fw primer, rev primer, template each.

PCR program

Step	temperature	time
(1)	98°C	30 s
(2)	98°C	10 s
	63°C	20 s
	72°C	20-25 s/ kb
(3)	72°C	2 min
(4)	4°C	hold

repeat step 2 25 cycles

Digest of pSB1C3 from BBa_K323005 (RFC10) with AgeI und NgoMIV (buffer 4, 2 h, 37 °C )

06.06.2013

Gel run of PCR 1-4 and digest pSB1C3

30 min at 120 V, afterwards 1 h at 170 V, poststaining for 45 min (15 µl GelRed auf 50 ml TAE)

PCR bands in wished size were cutted and frozen over night. Digest was not functional (only one band), new pSB1C3 is searched with RFC25 (–> Team Freiburg 2010 plasmid number 51).

07.06.2013

Repeating of PCR (for Gibson I)

Gel run of PCR

1 % agarose, 120 V, 60 min; Poststaining

cutting bands out

Gelextractionof PCRs from 6.6.+7.6.

Weigh gel sclice, put liquidated duplicates onto one column, implementation via standard protocol.

PCR1: 165,5 ng/µl
PCR2: 146,5 ng/µl
PCR3: 56,3 ng/µl
PCR4: 160,5 ng/µl

10.06.2013

Digest of pSB1C3 with new insert

No. 51 (BBa_K404152 pSB1C3_VP1up), insert size 411 bp, concentration 83 ng/µl; see 07.06.13,implementation see 06.06.

Gelextraction of digested BBa_K404152 pSB1C3_VP1up

83,5 ng/µl

Gibson I

cutted pSB1C3 + PCR1-4 analog to standard protocol

Retransformation of BBa_K404152 pSB1C3_VP1u and transformation of Gisbon from 10.06.

Use 4 µl Gibson mix for transformation and for retransformation 2 µl of 1:6 diluted BBa_K404152 pSB1C3_VP1up (implementation analog to standard protocol), plate on chloramphenicol dishes

11.06.2013

No colonies on Gibson plates, repeat of Gibson analog to 10.6., colonies of retrafo were minipreped after standard protocol

12.06.2013

Colonies of repeated Gibson were picked and plated out.

13.06.2013

clone 1: 137 ng/µl
clone 2: 158 ng/µl
clone 3: 112 ng/µl
clone 4: 104 ng/µl
clone 5: 141 ng/µl
clone 6: 38 ng/µl
clone 7: 62 ng/µl

Test digest of miniprep from 13.06.

Digest with NgoMIV, 10 µl total volume, clones 1-5 2 µl each, clones 6+7 3 µleach, wished bands: 2500 bp, 2200 bp, 1400 bp, 80 bp

Gel (1%) of test digest from 13.06.

no Poststaining (1 µl GelRed added into fluid agarose gel), 180 V, 1 h

Digest did not work, only one band is visible--> religated bb? (AgeI and NgoMIV are compatible), assumption: Gibson did not work because of spontaneous religation of digested backbone–> no Gibson reaction possible

New digest of Ba_K404152 pSB1C3_VP1up with phosphatase treatment

3 µg vector digested, after 105 minutes: addition of antarctic phosphatase reaction buffer (1-fold, conzentration is 10-fold)+ 1 µl Phosphatase + water up tp 30 µl

15 min at 37 °C
5 min at 60 °C

Test digest II of clne 7 of Gibson I (second trial) from 13.06.

digest with XbaI und AgeI in buffer 4, 1 h at 37 °C, wished sizes: 4119 bp (Cas9), 2076 bp (backbone), 287 bp

Gel (1%) of digest from 13.06. andof test digest II from 13.06.

From left to right: Marker diluted 3 µl, Marker undiluted 3 µl, test digest, digest, Marker diluted 1 µl, Marker undiluted 1 µl; test digest was not successful (only one band), bigger band of digest was cutted out and was being frozen

14.06.2013

Gel extraction of digested and phosphatase treated BBa_K404152 pSB1C3_VP1u from 13.06.

analogous to 09.06., 11 ng/µl

Repeat of Gibson I with new digested and phosphatase treated BBa_K404152 pSB1C3_VP1u from 13.06.

analogous to 11.06., afterwards 4 µl Gibson Mix were transformated

15.06.2013

Clones were picked from Gibson plates from 14.06.and plated on Chloramphenicol plates

16.06.2013

Minipreparations of picked colonies from 15.06.

17.08.2013

Test digest of minipreps from 16.06.

Digest with XbaI und AgeI in Cut smart buffer, 1,5 h at 37°C

Sequencing of clone 1.1 and 1.3 with oIG0002 (forward Primer of Cas9 till base 1085)

17.06.2013

Testdigest of Miniprep from 16.6.

Digested with XbaI and AgeI in Cut smart buffer, 1.5 h at 37°C

Sequencing of clone 1.1 and 1.3 with oIG0002 (forward Primer for Cas9 after 1085 bases)

24.06.2013

Digestion of pSB1C3_VP1_up (51)

Digestion was performed with AgeI-HF and NgoMIV in buffer 4 (1,5h 37°C) followed by CIP treatment (as on the 13th of June).

0,7% agarose gel (normal marker(1µl), digest, empty, pSB1C3 control(uncut), ready to use gene ruler marker (1µl)

Lower half of the band at 2000bp was cut out and purified with Roche PCR purification kit: 10ng/µl

July

02.07.2013

Gibson with digested pSB1C3 from 24.06.

Concentration of all PCRs and dilutions measured again (see below), volume for Gibson reaction calculated with excel sheet in dropbox. DNA directly added to Gibson Master Mix, 1h at 50°C, 4µl in trafo with competent cells (21.6.), inoculated in 500µl LB, plated on chloramphenicol plates. Negative control without PCR4.

Retrafo of pSB1C3_VP1up (51)

03.07.2013

Some colonies on Gibson plate, none on negative control. Six colonies inoculated for miniprepping. Retrafo of pSB1C3_VP1up (51): three colonies for minis inoculated.

Mini preps of pSB1C3_VP1up (51)

ini preps and test digest of Gibson from 2.7

Minis in freezer box (Standardisierung=St1-6). Test digest with AgeI-HF and XbaI in Cut-Smart buffer. Expected fragments: 4119bp (Cas9), 2076bp (pSB1C3)

19.07.2013

PCR of pSB1C3 as well as all single fragments 1-7 for Cas9 standardization

PCR was made analog to 5.6. For pSB1C3 60 °C were used as annealing temperature and pIG0037 and pIG0038 as primers. Two probes have been made: template amount of 10 pg and 1 ng (I/II).

PCR1: 839 bp, Primer oIG0022, oIG0036
PCR2: 330 bp, Primer oIG0035, oIG0028
PCR3: 1121 bp, Primer oIG0029, oIG0034
PCR4: 268 bp, Primer oIG0033,oIG0026
PCR5: 314 bp, Primer oIG0027, oIG0024
PCR6: 945 bp, Primer oIG0025, oIG0032
PCR7: 484 bp, Primer oIG0031, oIG0023

For pSB1C3: 2086 bp. PCR was frozen afterwards (o.n.)

20.07.2013

Gel 1 (big): M I I II II 3
Gel 2 (big): M 1 2 4 5
Gel 3 (small): M 6 7

Gelpicture could not be saved due to error of the machine. Size was not as excpected unless from amplification of the backbone pSB1C3. Gelextraction followed. Afterwards a test gel was made with 2 µl of the extracted band. Size was as excpected.

Fusion PCR of the PCR products 1-7

Probe 1: PCR 1-7
Probe 2: PCR 1-3
Probe 3: PCR 4-7

PCR I: Annealing temperature was performed after the lowest overlapping temperature of the primer (1+3: 50°C, 2: 54°C) Elongation time was performed after the longest fragment size of the several PCR fragments to be fused (1+2: 28 sec, 3: 24 sec; run all with 28 sec). PCR II: primer added in each of the probes (oIG0022+oIG0023 for 1, oIG0022+oIG0034 for 2, oIG0033+oIG0023 for 3) Elongation time was performed after the product size (1: 1 min 44 sec , 2: 57 sec, 3: 50 sec, 2+3 have been run on 57 sec)

4162 bp for 1
2290 bp for 2
2011 bp for 3

Repetition of PCR of amplification of pSB1C3

1 ng has been used as template volume, template was pSB1C3 (RFC10–> Probe I) and pSB1C3 (RFC25–> Probe *). PCRs were frozen o.n.

21.07.2013

Agarose Gel from PCR of 20th of July

0.7 % Agarose Gel has been run

Gelextraction from PCRs of 20th of July

Fusion 1: 26 ng /µl
Fusion 2: 40 ng /µl
Fusion 3: 62 ng /µl
PCR*: 8 ng /µl.

22.07.2013

Fusion PCR of Fusion-PCR 2+3 of the 20th of July

PCR 1: Elongationtime 1 min 2 sec, 1 µl of each PCR-Product, Annealing temperature: 56 °C.

PCR 2: Elongationtime 1 min 44 sec, Annealing temperature: 63 °C, primer added: oIG0022+oIG0023.

FPCR of amplification PCR of the 20th of July (of pSB1C3_RFC 25 backbone)

2 µl template, 52 sec elongation, 60 °C annealing, primer oIG0037+38

Gibson assembly of PCR products from the 20th of July (of pSB1C3_RFC 25 backbone)

Gibson 1 (no. 1): Fusion PCR product 1 (from PCR 1-7 from 19.7.)+ PCR of pSB1C3_RFC25 bb (*)
Gibson 2 (no. 2/3: Fusion PCR Product 2 (from PCR 1-3 from 19.07.)+Fusion PCR Product 3 (from PCR 3-7 from 19.07.)+ PCR of pSB1C3_RFC25 bb (*)
Gibson 3 (no. -): Fusion PCR Product 3 (from PCR 3-7 from 19.07.)+ PCR of pSB1C3_RFC25 bb (*)

Gibson 1: 1 µl fusion 1-7 (no 1), 3.1 µl bb, 0.9 water
Gibson 2: 0.6 µl fusion 1-3 (no 2), 0.4 µl fusion 3-7 (no 2), 3.1 µl bb, 0.9 water
Gibson 3: 0.4 µl fusion 3-7 (no 2), 3.1 µl bb, 1.5 water

Trafo of Gibson assembly from the 22th of July

put TOP10 on Chloramphenicol o.n.

Gelrun of the PCRs from the 22th of July

0.7 % Agarose gel, loading scheme: M, *(3 µl),*(all), *(all), *(3 µl), PCR 1(3 µl), PCR 1(all), empty, Fusion 2/3 (3 µl), Fusion 2/3 (all)

Expected size: * (2086 bp), PCR1 (4162 bp), Fusion2/3 (4162 bp)

fragments were cutted out and frozen o.n.

23.07.2013

PCR over Fusion PCR product 1-7 was performed

primer oIG0022+23, elongation time 1 min 44, product was frozen o.n.

24.07.2013

Gelrun of PCR from 23.7. with 0.7 % Agarose and 120 V

From left two right: Marker 2log from NEB, 5 µl PCRI, 56 µl PCRI,56 µl PCRII, 5 µl PCRI, 1 µl.

Ciped pSB1C3_RFC25 bands were cutted out and gelextracted over one column (17 ng/µl, 30 µl total volume.

Digest of Fusion PCR1-7 and pSB1C3_Vp1Up_RFC25 with EcoRIHF and SpeIHF

For both 40 µl as total volume:

pSB1C3: 23 µl DNA [2,5 µg, conc.:111 ng/µl], Eco and Spe 1 µl each , buffer cut smart 4 µl, up to 40 µl with water (11 ml)

Fusion 1-7 [digest all of the gelectracted product]: 25 µl [425 ng, 17 ng/µl], Eco and Spe 0.5 µl each, , buffer cut smart 4 µl, 10 µl water up to 40 µl

2 h, 37 °C

PCR of Fusion PCR1-7 and PCR over non-nickase Cas9 (plasmid 2004)

PCR over non-nickase PCR with oIG27+24, elongation time 8 sec [plasmid 1 µl of 1:10 dilution, because of high concentration of plasmid–> midiprep)

PCR of Fusion PCR1-7 see 23.07.

Gelrun of digest from 24.07.

excpected size: 2051 bp for pSB1C3 [insert left: 454 bp], 4144 bp for Fusion 1-7

We run gel very long for cutting out digested backbone using lower band for later ligation.

Because we cutted out Fusion 1-7 digested without being able to diverge the cutted and uncutted PCR product, 1:1 ratio is used for ligation

Gelextraction of the two digested fragments from 24.07.(pSB1C3_Vp1up and Fusion1-7) pSB1C3_Vp1up: 28 ng/µl Fusion1-7: 7 ng/µl

Ligation of the two digested fragments from 24.07.(pSB1C3_Vp1up and Fusion1-7)

We have been used Cas9 as bb and pSB1C3 as insert with an 1:1 ratio.

20 µl total volume, 15 min, 22 °C, 1 µl T4 ligase, 2 µl T4 ligase buffer, 30ng from Cas9 [4 µl], 15 ng of pSB1C3_RFC25 [0.5 µl]

Transformation of ligation from 24.07.(pSB1C3_Vp1up and Fusion 1-7)

2.5 µl of ligation result to 25 µl TOP10

Gelrun of PCR from 24.07.

excpected size: 4162 bp for PCR1-7, 314 bp for PCR over non-nickase Cas9 (plasmid 2004)

PCR over fusion PCR1-7 did not work, is repeated

right picture: marker log2, PCR over p2004 with oIG0027+24, fragments were cut out and gelextracted (32,1 ng/µl)

25.07.2013

Result of Ligation from 23.07

One clone was visible. Clone was picked and given into 3 ml LB over night and icubated with 37° C.

PCR of Fusion PCR 1-7 on 23.07.13

Elongation time 1 min 44 (Rest like above)

Fusion PCR 1-7 with PCR Fragment 5 without Nickase (PCR Produkt from 23.07.13

Fusion PCR approach like above, except for amount of PCR Products: 3 µl of PCR Product 4 and 1 µl of the Rest. PCR Product 4 is now empty. Repeat PCR 4

26.07

Result of Sequencing of Fusion PCR 1-7

Results were positive, Primers were able to bind at expected basepairs of Cas9. → Fusion PCR works!

Minipreps of Transformation from 24.07

Concentration: 76 ng/µl

Test digest of Transformation from 24.07

Digested with XbaI and AgeI HF

29.07

Digest of pSB1C3 RFC 10 and Fusion PCR Product 1-7 * (Double Mutant, without Nickase)

Fusion PCR was digested with EcoRI and SpeI; pSB1C3 was cutted with XbaI and AgeI HF (with phosphatase treatment)

Ligation of Fusion PCR Product for Cas9 without Nickase and pSB1c3 RFC10

BB to Insert ratio was set 1:1, ligation was incubated 15 minutes at room temperature

30.07

Two colonies were visible. They were plated out for minipreps.

31.07

Colony 1: 344,9 ng/µl
Colony 2: 254,0 ng/µl

Test digest of Transformation from 30.07

cutted with XbaI and AgI HF

August

01.08

Sequencing result

Sequencing result of Cas9 in pSB1C3 looks good. The one mutation that can possibly be seen with oIG6017 is present. Cas9 is now in the shipping backbone. More primers were send to GATC in order to sequence the whole Cas9 (oIG0001, oIG0002, oIG0003, oIG0004, oIG0005).

02.08

Sequencing results

Cas9 seems to be mutated completely (Primers oIG0001, oIG0002, oIG0003, oIG0004, oIG0005).

16.08.2013

Transformation of CMV-Promotor(BBa_K747096) and AAV2-NLS (BBa_K404153)

CMV-Promotor plasmid was extracted from the distribution-kit of 2013 (plate 1, 21P). E. coli were plated out on Chloramphenicol containing plates.

Oligo annealing of oIG0047 and oIG0048 (HA-tag)

Oligo 1 (100 µM)	50 µl
Oligo 1 (100 µM)	50 µl
water	150 µl

Incubate at 98 C for 4 minutes and cool down in turned off heating block for 3 hours.

PCR of BGH terminator

BGH was amplified from pGR2 with the primers oIG0049 + oIG0050.

Annealing temperature was 62 °C, elongation time was set to 7 seconds (wished product size: 242 bp).

Digest of pIG001 with NgoMIV and SpeI

2.5 µg of plasmid were digested with NgoMIV and SpeI HF in cut smart buffer for 2 hours at 37 °C.

Expected band size 2100 bp (bb) and 4121 (Cas9 Insert)

Gelrun of digest and PCR from 16.08.

PCR and digest were run on a 1% agarose gel and disered band sizes were cutted out.

Gelrextraction from digest and PCR bands from 16.08.

BGH: 47 ng/µl
pIG001 cutted with NgoMIV and SpeI: 18 ng/µl

Ligation of pIG001 cutted with NgoMIV and SpeI and HA-oligo

HA-oligo was diluted 1:100 and concentration measured with nanodrop (7 ng/ µl).

50 ng of bb was used (2.8 µl of cutted pIG001)and an 3 times excess of Insert (17.3 ng, 2.5 µl of HA-oligo)

[50ng/bp vector*3=ng insert/bp insert].

Ligation was incubated 40 minutes at 22 °C and afterwards transformed.

Gibson assembly of pIG001 cutted with NgoMIV and SpeI and BGH-Terminator

1:4 ratio of bb:insert was used.

For DNA mix 1.4 µl of cutted pIG001, 0.25 µl of BGH terminator and 3.35 µl water were used.

Gibson mastermix was added to the DNA mix, incubated 1 hour at 50 °C and afterwards transformated.

Transformation of ligation and Gibson from 16.08.

Top10 cells were plated out in chloramphenicol containing plates.

Start standardisation of VP16 and KRAB and SV40 Promoter

PCR was performed at 60°C annealing temp:

oIG0042/0041 –> KRAB ( 445bp)
oIG0040/0039 –> VP16 (451bp)
oIG0043/0044 –> pSV40 (411bp)

Results:

PCR products were band isolated and a gel extraction was performed.

Gibson of VP16 KRAB and SV40 into pSB1C3

Gibson was performed using the following approach:

17.08.2013

Picking and plating of colonies from the transformation of 16.08.

Continue standardisation of VP16 and KRAB and pSV40

Colonie PCRs were performed using 60°C annealing temp and oIG6017 and oIG6018 after standard protocoll.

Results:

The positive colonies (upper row) were picked for mini prep and one of each was send for sequencing.

18.08.2013

Miniprep of plated colonies from the 17.08.

yield was between 30 and 50 ng/µl

19.08.2013

Test digest of the miniprep from 18.08.

HA-tag (4 minipreps): cut with AgeI (for gel NgoMIV and SpeI cut pIG0001 was used as control)
CMV (580 bp): cut with XbaRI and SpeI HF
BGH (4 minipreps, 222 bp): cut with AgeI HF and Eco HF
NLS(21 bp): only sent to sequencing

Gel run of test digest from 19.08.

From left to right: Marker 2 log, HA (4x), HA Kontrolle, CMV, BGH (4x), Marker 2 log

BGH terminator seemd not to have worked. HA, NLS and CMV were sent to sequencing. As sequencing primer pIG6017 has been used (fv primer binding around 100 bp before the insert).

20.08.13

Sequencing results

All three parts are fully sequenced without mistakes.

Digest of the parts NLS, HA-Tag, CMV- & SV40-promoter and Cas9

ingredient	volume
plasmid	1 µg
restriction enzyme	each 1 µl
NEB buffer CutSmart	3.5 µl
dH₂O	up to 35 µl

Incabation at 37 °C for 2 h.

plasmid	restriction enzymes
pIG001 (Cas9)	NgoMIV & PstI HF
NLS	AgeI HF & PstI HF
HA	EcoRI HF & XbaI
CMV	EcoRI HF & SpeI HF
SV40	EcoRI HF & SpeI HF

Gel run

From left to right: Marker (1 kb Roth); SV40; HA; Cas; NLS; CMV

Expected bands: ~ 4kb for Cas9; ~ 2kb for NLS and HA (incl. Bb); 580 bp for CMV; 340 bp for SV40.

From left to right: Marker (1 kb Roth); SV40; HA; Cas; NLS; CMV

Gel extraction

DNA were purified using High Pure Plasmid Isolation Kit of Roche.

Changes to the protocol:

incubation at 56 °C for 10 min for gel dissolving
elution with dH₂O (incubation at 50 °C for 4 min before centrifugation)

Yield: 5 ng/µl for CMV and SV40; ~ 12 ng/µl for Cas9 and HA; 61 ng/µl for NLS

Ligation

ingredient	amount
NLS (with Bb)	0.5 µl
Cas9	14.9 µl
T4-Ligase	1 µl
T4-Ligase buffer	2 µl
dH₂O	up to 20 µl

ingredient	amount
HA (with Bb)	3 µl
CMV / SV40	6 / 3.5 µl
T4-Ligase	1 µl
T4-Ligase buffer	2 µl
dH₂O	up to 20 µl

Incubation at 22 °C for 30 min.

Transformation was made from the ligation of 20.08. and retrafo of standardized HA, NLS, CMV

Colony PCR of the BGH Gibson plate from 16.08

Colony PCR was made analog to standard protocol. As primer pIG6017 and pIG6018 are used. 12 clonies had been screened. Excpected size of product: 550 bp

Loading scheme: M,colony 1-12, positive control (standardized NLS with 349 bp product size) Clone 6 has been plated out over night.

21.08.13

Minipreps from the plated BGH/retrafos from 20.08.

yield around 65 ng/µl; BGH Trafo was send to sequencing (with pIG6017)

Colony PCR of clones of the ligation from 20.08.

12 colonies were tested for CMV-HA, SV40-HA, NLS-Cas9 with oIG6017+18.

Colony PCR of CMV-HA (excpected size: 950 bp):

loading scheme: M, 1-12, positive control (standardized NLS); Clone 4 was picked and plated out.

Colony PCR of NLS-Cas9 (excpected size: 4.5 kbp):

loading scheme: M, 1-12, M; Clone 3 was picked and plated out.

Colony PCR of SV40-HA (excpected size: 700 bp):

loading scheme: M, 1-12, M; Clone 11 was picked and plated out.

Oligo annealing of linker fv and linker rev (7 AS, oIG0051+52)

Oligo 1 (100 µM)	50 µl
Oligo 1 (100 µM)	50 µl
water	150 µl

Incubate at 98 C for 4 minutes and cool down in turned off heating block o.n.