Synthetic miRNA Kit

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⇐ Synthetic miR Kit

21/08/2010

Gel1

Gel2

• all primers for the synthetic miR construction Kit were diluted to a 100 uM concentration
• PCR reaction for construction of the following parts were set up:

• CMV (reverse complementary); template: pCMV_BBB, primer: CMV_rc_BBB_fw/rev
• BGH terminator; template: pcDNA5/TO/FRT/miRsAg, primer: BGH_pA_fw/rev
• SV40 promoter (reverse complementary); template: pSiCheck-2, primer: SV40_rc_BBB_fw/rev
• RSV5' region; template: pBS/U6-GFP, primer: RSV_fw/RSV_rev_midcomp
• RSV3' region; template: pBS/U6-GFP, primer: RSV_rev/RSV_fw_midcomp
• RSV5' region (reverse complementary); template: pBS/U6-GFP, primer: RSV_rc_rev/RSV_rev_midcomp
• RSV3' region (reverse complementary); template: pBS/U6-GFP, primer: RSV_rc_fw/RSV_fw_midcomp

The PCRs were set up according to the following protocol:

• 0.5 ul (= 50 ng) Template DNA
• 10 ul Hifi-Buffer
• 0.5 ul each Primer
• 1 ul (= 2.5 u) Hifi-Polymerase
• 37.5 ul water

The PCR was run according to the following protocol:
................................................

95 °C/ 5 min

................................................

94 °C/ 15 sec

57 °C/ 1 min

72 °C/ 1 min

................................................ (35x)

72 °C/ 10 min

................................................

4 °C/ forever

................................................

• PCR reaction for the construction of the linear standard plasmids was set up as follows:

• 1 ul (10 ng) template DNA pSB1A3/pSB1K3/pSB1C3, cut EcoRI/PstI/DpnI
• 0.5 ul of each primer SB-prep-3p/SB-prep-2Ea
• 10 ul of Hifi Buffer
• 2 ul of Hifi Polymerase
• 36 ul water

The PCR was performed according to the following protocol:
................................................

95 °C/ 5 min

................................................

94 °C/ 15 sec

55 °C/ 1 min

72 °C/ 2.5 min

................................................ (35x)

72 °C/ 10 min

................................................

4 °C/ forever

................................................
The result of the PCRs was analyzed on a 1% agarose gel, run for 1 h @ 100 V (Gel1).
As the result of the first pSB vector PCRs was not ideal due to strong side products, the PCR was repeated in 6 replicates according to the following protocol:

• 25 ul Phusion PCR MasterMix
• 1 ul template DNA (pSB1C3, digested with PstI/EcoRI/DpnI)
• 0.5 ul of each primer SB-prep-3p/SB-prep-2Ea
• add water to final volume of 50 ul

The 2.1 kb band was afterwards cut out from the gel and a gel extraction was applied for purification of the DNA (Qiagen Gel Extraction Kit).
PCR was performed, applying the standard protocol (annealing temperature 60 °C). 5 ul of PCR product were analyzed on an agarose gel, run for 1 h @ 100 V.

22/08/2010

• Fusion-PCR for the construction of the whole RSV promoter fragment with BBB prefix and suffix:

• 1 ul (50 ng) of RSV5' template (or RSV5' (reverse complementary))
• 1 ul (10 ng) of RSV3' template (or RSV3' (reverse complementary))
• 10 ul buffer
• 1 ul Hifi Polymerase (2.5 u)
• 36 ul water

The PCR was performed according to the following protocol:
................................................

95 °C/ 5 min

................................................

94 °C/ 15 sec

58°C/ 1 min

72 °C/ 1 min

................................................ (35x)

72 °C/ 10 min

................................................

4 °C/ forever

................................................

• PCR for the construction of linear vector, the PCR was further optimized by applying again the Hifi and the Phusion PCR protocol from the previous day, but using an annealing temperature of 64 °C that time.

The PCR for both fragments was analyzed on a 1 % agarose gel, run for 1 h @100 V (Gel1) and PCR purified applying a Qiagen PCR purification Kit.

• Digestion of PCR product and Vectors

1 ug of each vector PCR and insert PCR product was digested with EcoRI/PstI according to the NEB standard digestion protocol (10 u Enzyme, final volume 50 ul). After heat inactivation (20 min @ 80 °C), 2 ul (2 u) of shrimp acidic phosphatase were added to the vector digestions and incubated for 30 min @ 37 °C. After the digestion, a purification was performed using a Qiagen PCR purification kit.

• Ligation

Ligations were set up using the Fermentase Ligation Kit. The following vector and insert ratios were used:
Vector 1 (Gel Extracted from previous day) and vector 3 (Phusion PCR product, PCR purified); 4 ul (40 ng):

• CMV(reverse complementary): 45 ng
• BGH terminator: 27 ng
• SV40(reverse complementary): 37 ng
• RSV: 50 ng
• RSV(reverse complementary): 50 ng

Vector 2 (Hifi PCR product, PCR purified); 4 ul (80 ng):

• CMV(reverse complementary): 90 ng
• BGH terminator: 55 ng
• SV40(reverse complementary): 75 ng
• RSV: 100 ng
• RSV(reverse complementary): 100 ng

Furthermore, negative controls were set up for each vector. Transformation in E. coli Top10 cells were performed according to the standard transformation protocol.

23/08/2010

gel1

Gel2

Colony-PCR of the colonies obtained on the agar-plates (cloning previous day) was performed according to the following protocol:

• 12.5 ul Phusion MasterMix
• 0.25 ul of each primer
• 12 ul of water
• colony picked from agar-plate

2 colonies were picked for each construct. The PCR was performed according to the standard Phusion PCR protocol. In parallel, a MiniPrep Culture for each colony was inocculated.
PCR for the construction of Luc2, hRluc, CMV, SV40 promoter, SV40 terminator and the BGH-Terminator. Therefor, PCR reactions were set up according to the following protocol:

• 10 ul Hifi Buffer
• 0.5 ul of each primer
• 1 ul (50 ng) of each template
• 38 ul of water

The combination of templetes and primers were as follows for the mentioned constructs:

• CMV: primers CMV_BBB_fw/rev; template: CMV_BBB (Team Heidelberg 2009)
• Luc2: primers: Luc2_ter_BBB_fw/Luc2_ter_BamHI_BBB_rev; template: pGL4.23 (Promega)
• hRluc: primers: hRluc_ter_BBB_fw/hRluc_ter_BBB_rev; template: pSiCheck-2 (Promega)
• SV40 promoter: primer: SV40_BBB_fw/rev; template: pSiCheck-2 (Promega)
• SV40 terminator: primer: SV40_term_fw/rev; template: pGL4.23 (Promega)
• BGH terminator(reverse complementary): primer: BGH_pA_BamHI_rc_fw/BGH_pA_rc_rev

PCR was performed according to the standard Hifi-Protocol (annealing temperature 57 °C)

Gel2

24/08/2010

Gel1

Gel2

Gel3

• Amplification of the CMV Promoter as well as the amplification of pSB1A3/C3/K3 standard plasmid backbones was performed via Phusion or Hifi-PCR according to the standard protocol (annealing temperature 61 °C).

The PCR product was run on a Gel for 1 h @ 100 V and analyzed an a 1 % agarose gel (gel 1).

• Another colony-PCR of the colonies obtained on the agar-plates (cloning two days before) was performed according to the following protocol:

• 12.5 ul Phusion MasterMix
• 0.25 ul of each primer
• 12 ul of water
• colony picked from agar-plate

Two colonies each were picked from plate (1) and (2) and four colonies of plate (3) for each construct, respectively. The PCR was performed according to the standard Phusion PCR protocol. In parallel, a MiniPrep Culture for each colony was inocculated. The result of the colony-PCR was analyzed on a 1 % agarose gel, run for 1 h @ 100 V (gel2).

• a PCR for the construction of synthetic microRNAs and microRNA binding sites was perfomed. For the construction of binding sites, single stranded oligos were ordered and second strand synthesis was performed using the BBB_suffix_reverse primer. The protocol was as follows:

• 1 ul microRNA binding site oligo
• 0.5 ul BBB_suffix_reverse primer
• 23.5 ul water
• 25 ul Phusion MasterMix

The binding site oligos for the following microRNA binding sites were used:

• hsa-miR-375 (perfect, 9-12 mut, 9-22 mut)
• hsa-miR-376a (perfect, 9-12 mut, 9-22 mut)
• hsa-miR-122 (perfect, 9-12 mut, 9-22 mut)
• shRNA-6 (perfect, 9-12 mut, 9-22 mut)
• shRNA-7 (perfect, 9-12 mut, 9-22 mut)
• shRNA-8 (perfect, 9-12 mut, 9-22 mut)
• shRNA-9 (perfect, 9-12 mut, 9-22 mut)
• shRNA-10 (perfect, 9-12 mut, 9-22 mut)

• PCR for the construction of synthetic shRNAs was performed. Therefore, the pcDNA5/TO/FRT containing the shRNA miRsAg as insert was used as template. For the shRNAs 1,2,3,5,6,7,8,9,10 the synthesis of two intermediate fragments were performed. The 5' fragment was amplified by using the primer shRNA_AflI_BBb_fw and the reverse oligos of the certain shRNA gene. The 3' framents were amplified by using the primer shRNA_HindIII_BBb_rev and the forward primer of the certain shRNA gene. The forward and reverse primer of the shRNA gene introduce the guiding and passanger with certain specificities into the shRNA constuct. The miRsAg is an miR-122 like shRNA and uses the backbone of miR-122, but another guiding and passanger strand. The PCR was set up according to the standard protocol (annealing temperature: 58 °C).

The results of the PCR were analyzed on a 2 % agarose gel, run for 30 min @ 100 V (gel 3)).

25/08/2010

The constructs were tested in a digestion with NotI using the following protocol:

• 4.0 ul template
• 2.5 ul NEB buffer 3
• 2.5 ul BSA
• 0.5 ul NotI (NEB)
• add 25 ul

For RSV, RSV(reverse complementary), BGH(forward) und SV40 promoter (reverse complementary) positives bands were found and samples were send for sequencing.

• Cloning of constructs BBb_CMV, Luc2_ter, hRluc, sv40(forward) and BHG(forward):
  

Digestion of pSB1C3 and the PCR products of the mentioned constructs with EcoRI/PstI (1 ug each) according to the standard digestion protocol. Subsequent purification using a nucleaotide removal kit and ligation overnight at 16 °C.

26/08/2010

• The sequencing result for the samples send for sequencing on the privious day was checked. The results were positive for all samples, sequences were accurate.
• Transformation of the ligation done on the previous day according to the standard transformation protocol.

27/08/2010

gel1‎

gel2‎

• PCR was carried out for the following parts, using the standard HiFi protocol:

1. Backbone amplification using primers backbone_AvrII_fw and backbone_AvrII_rev

2. CMV-TetO2: CMV_TetO2_BBB_fw, CMV_TetO2_BBB_rev

3. Tet-repressor: TetRepressor (from pcDNA6)fw, TetRepressor (from pcDNA6)rew

4. FRT site: FRT_BBB_fw, FRT_BBB_rev

• Colony PCR was carried out (according to the standard PhusionMix protocol, annealing temp. 57 C) for CMV, Luc2, hRluc, SV40, SV40 ter and BGH-1 rc. Only one sample for hRluc and one for SV40 ter showed a possible positive result (gel1).

• The three standard plasmids (pBS_1A3, pBS_1C3, pBS_1K3) were digested with EcoRI and PstI according to the following protocol:

5 ul of each plasmid (conc. = ?)

3 ul EcoRI buffer

3 ul BSA

0.5 ul of each enzyme

18 ul nuclease-free water

Incubatation: 37 C, 1hr

Heat inactivation: 80 C, 20 min

Then DpnI was added (0.5 ul), and the tubes were incubated at 37 C for 1 hr, then heat-inactivation at 80 C for 20 min.

5 ul of the product were loaded on a 1% agarose gel and analyzed (gel1).

28/08/2010

• 6 more colonies from the cloning done on the 25th/26th were picked and colony PCR was performed according to the standard colony-PCR protocol. In parallel, Minipreps of each colony were inocculated. The results of the colony-PCR ist shown in the gelpictures.
• shRNA construction via PCR; Template: 50 ng of first PCR product; Standard PCR protocol using an annealing temperature of 60 °C.

29/08/2010

gel1

gel2

• Vector pcDNA_BBbCMV, PCR product Luc2_ter and shRNA(1-10) with EcoRI/PstI (1 ug each). The BBb_CMV was cut out from the gel (~ 700 bp band) and gel purified (gel1). The PCR product Luc2_ter was purified by applying a nucleotide removal kit.
• Ligation reactions were set up using pSB1C3 (amplified using Phusion HF Mastermix (1) or High Fidelity Polymerase (2) cut EcoRI/PstI) (100 ng) and 100 ng CMV, 40 ng shRNA or 300 ng Luc2 constructs. For shRNA6 and CMV the cloning was done in 2 replicates (using either the pSB1C3 amplified via HF Mastermix or High Fidelity Qiagen Kit). The ligation was performed according to the fermentas ligation kit protocol and incubated for 1 h @ room temperature. Afterwards, 10 ul were transformed into Top10 cells.

30/08/2010

Gel1

Gel2

Gel3

• The ligation result from the previous days' cloning was analyzed via colony PCR (gel 1-3) and all colonies were inocculated (LB cultures) The construct numbers indicate the number of the clone picked from the agar plate. If cloning was performed in Replicates, the first number indicates the plate (i.e. CMV3) and the second number the clone. As seen in gelpicture 1 the clone CMV3.1 and on gelpicture 3 the clones CMV 2,3,12 and 16 all show the right band length of ~1000 bp. On gelpicture 2 the shRNA10.1 clone shows the right band as well as the Luc2.1.5 clone for the Luc2 luciferase.

31/08/2010

Gel1

Gel2

• The LB cultures from the previous day were inocculated and mini prepped. Afterward, a test digestion of all constructs with NotI was performed (gel1) and loaded on a 1 % agarose gel run for 35 min @ 100 V. The samples CMV2, CMV3 (23), CMV3.1, Luc2.1 and sh10.1 showed the right bands on the gel and samples were send for sequencing. All sequencing results showed, that the constructs were right.
• pSMB_Measure backbone (igem team HD 2009 measruement standard) was amplified using the ClaI/XhoI or AvrII primers and performing a standard Phusion HF Mastermix PCR protocol (annealing temperature 61 °C. The PCR result was loaded on the agarose gel 1 for control (BcA = AvrII cutting sites, BcB = XhoI/ClaI cutting sites). The PCR products of the used vectors pSB1C3 and the hRluc and Luc2 PCR fragments used in this and subsequent clonings was also loaded on the gel for controlling purposes.
• Digestion of the pSB1C3 vector amplified via Phusion HF PCR Mastermix (annealing temperature 61 °C) with EcoRI/PstI for 1 h @ 37 °C. After heat inactivation, 1 ul of DpnI was added to the restriction mix an digestion performed for another hour. The product was SAP (dephosphorylation) or not SAP treated this time. Furthermore, the hRluc, Luc2, CMV_TetO2, sv40 and FRT site PCR products (see 08/27/2010) were digested EcoRI/PstI as well.
• Ligation: each PCR fragment was ligated into digested vector backbone pSB1C3. 50 ng of the non-treated and 100 ng of SAP treated vector were used in the ligation reactions. Amount of insert was calculated to be 2 fold (large fragment Luc2 and hRluc) or 3-5 fold of the molar vector amount. Ligation was performed for 1.5 h @ room temperature. The ligation was analyzed on a 1 % agarose gel, run for 35 min @ 100 V. The non-treated vector is indicated via number 1 (i.e. Luc2.1) the SAP-treated vector with nr. 2 (i.e. Luc2.2). As control, the digested insert fragment PCR products and digested vectors were loaded on the gel as well. For hRluc, Luc2 and CMV_TetO2 correct product bands are clearly visible. For sv40, that is not the case. The FRT site is to short, to be able to distinguish clearly between vector fragment and ligated Vector with Insert. Subsequently, a transformation of all 10 ligation reactions into Top10 cells was performed according to the standard transformation protocol.

⇓ September

⇐ Main Page

⇐ Synthetic miR Kit

⇑ August

01/09/2010

Gel 100901-1

Gel 100901-2

• 8 colonies of each plate (see previous day) were picked and colony-PCR was performed. Furthermore, the colonies were used for inocculation of LB miniprep cultures. The numbers behind the construct name refer to vector type (1 = not SAP treated, 2 = dephosphorylated using SAP; see previous day). For hRluc (1300 bp band)), Luc2 (2300 bp band), CMV_TetO (1000 bp band) and the FRT (500 bp band) site, positive colonies could be observed (gel 100901-1 and 100901-2).
• PCR for the construction of the standardized Tet Repressor was performed. The PCR was set up using the Phusion HF MasterMix and standard PCR protocol was applied (annealing temperature 59 °C, 2 replicates). The result of the PCR was analyzed on a 1 % agarose gel (gel 100901-2, lower right side) and the right 700 bp band was clearly visible.
• Digestion of the TetR fragment with EcoRI/PstI was performed according to the standard digestion protocol and the fragment was subsequently ligated into pSB1C3 (SAP treated or non-treated). The ligation mix was transformed into Top10 cells.

02/09/2010

Gel 100902-1

• 8 colonies were picked from plates 1 and 4 colonies from plates 2 from the previous days cloning and analyzed via colony PCR performed according to the PCR standard protocol. The results of the colony PCR were analyzed on a 1 % agarose gel run for 1 h @ 100 V (gel 100902-1 and 100902-2). The following colonies were afterwards inocculated (LB miniprep cultures):

• TetR (1.4 and 1.6)
• shRNA6 (1.1, 1.4 and 1.8)
• shRNA7 (1.1, 1.5 and 1.6)
• shRNA8 (1.1, 1,2, 1,3)
• shRNA9 (1.2)

• The cultures inocculated the day before were mini prepped by applying a Qiagen Miniprep Kit. Afterwards, a test digestion with NotI was performed and the result was analyzed on a 1 % agarose gel, run for 35 min @ 100 V (gel 100902-3). According to the test digestion result, the following samples were send for sequencing @ GATC:

• CMV_TetO2 (.4, 1.6)
• FRT (1.1, 1.3)
• Luc2 (1.4, 1.6)
• hRluc (1.1, 1.3, 1.4)

Gel 100902-2

Gel 100902-3

03/09/2010

Gel 100903-1

Gel 100903-2

• Test digestion with NotI of the cloned constructs on the previous day (gel 100903-1). For the TetR constructs the expected 600 bp band and for the shRNA constructs, the expected 200 bp band were clearly visible. For each construct, one sample was send for sequencing. Sequencing results were correct for all samples send for sequencing.
• Purification of single binding sites against shRNA6-10, miR-375/376/122. For each target, the perfect, 9-12 and 9-22 randomized binding sites were purified and analyzed on an agarose gel (100903-2). All bands were clean, with just a slight amount of side product for the 9-22 randomized binding sites.

05/09/2010

• transformation of final Kit constructs F1 - F16 into E. coli Top10 cells

06/09/2010

• inocculation of miniprep LB cultures for the constructs F1 - F16 from single colonies from the plates prepared the previous day
• digestion of PCR amplified vector backbone pSB1A3 with EcoRI/PstI; after subsequent heat inactivation, digestion with DpnI was performed and the vector was purified by applying a nucleotide removal kit.

07/09/2010

Gel 100907-1: Ligation Control. Numbers indicate the F-Construct numbers of the parts assembled

• Miniprep of constructs F1 - F16 using a Qiagen Miniprep Kit
• Digestion of 500 ng of each construct with the following enzymes

• F2, F3, F5 with EcoRI/NheI
• F8, F1, F15, F11, F12, F13, F14, F7 with PstI/SpeI

• Ligation of the follwing constructs into pSB1A3 via standard 3A assembly

• F3/F15, F2/F1, F2/F8, F3/F1, F2/F15, F5/F7, F5/F11, F5/F12, F5/F13, F5/F14

• Digestion of 500 ng F6 with XbaI; purification via nucleotide romoval kit
• Annealing of XbaI mutation oligo according to the following protocol

• prepare mixtrure of oligos (5 ul @ 100 mM each) in NEB buffer 2 (volume 50 ul)
• heat up to 95 °C in heating block; cool down to room temperature slowly
• incubate on ice for 10 min

• 0.5, 1 and 3 ul of the annealed oligos were then ligated into 50 ng of pre-digested F6 construct;

All Ligations were controlled by loading 10 ul of ligation reactiong onto a 1 % agarose gel (100907-1), run for 35 min @ 100 V

08/09/2010

Gel 100908-1: Colony-PCR.

• Colony PCR of the previous days' cloning and miniprep cultures were inocculated in parallel; The result of the PCR was analyzed on a 1 % agarsoe gel (100908-1) run for 35 min @ 100 V.

09/09/2010

Gel 100909-1

Gel 100909-2

• Miniprep of the cultures inocculated on the previous day followed by test digestion of 300 ng of each constructs with NotI (gel 100909-1). The positive samples were send for sequencing @ GATC. The gel looks weird due to use of 0.05 x TAE buffer instead of 0.5 x.
• The nine Luc2-SV40 constructs were with XbaI and NheI (negative testing), and XhoI and PstI (positive testing) for the induced mutation. The reaction was set up according to the standard protocol, and then the samples were visualized on a 1% agarose gel (gel 100909-2).

10/09/2010

• PCR of SV40 Terminator with BamHI site according to standard PCR protocol (annealing @ 58 °C)
• Assembly of the following constructs, again via 3A standard assembly.

• F6/F1, F6/F8, F10/F4, F5/F1, F16/SV40, F5/F8
  

Subsequent Transformation into DH5alpha cells

11/09/2010

Gel 100911-1

Gel 100911-2

• Colonies were picked from the plates from the previous days' cloning and a colony PCR was performed according to the standard protocol. In parallel LB miniprep cultures were inocculated for each colony (gel 100911-1 and 100911-2).

12/09/2010

Gel 100912-1

Gel 100912-2

• Minipreps were done by applying a Qiagen Miniprep Kit for the positive candidates picked on the previous day. Test digestion was performed by digesting ~ 250 ng of each construct with SpeI/NheI and the result was analyzed on a 1 % agarose gel (100912-1 and100912-2) run for 40 min @ 100 V. For all the constructs we got the right band despite construct F10/F4.3. Positive constructs were send for sequencing @ GATC.

13/09/2010

• digestion of the following parts:

• F4 (E/N), F5 (S/P), R5 (E/N), R6 (E/N), R1 (S/P), R2 (S/P), R3 (S/P), R4 (S/P), R9 (E/P), R10 (E/P); after heat inactivation subsequent purification using a nucleotide removal kit (elution volume 20 ul).

• ligation of the following parts (50 ng of vector, equal amounts of parts)

• F4/F5 into pSB1A3 (precut E/P)
• R5 with either R1, R2, R3 or R4 into pSB1C3 (precut E/P)
• R6 with either R1, R2, R3 or R4 into pSB1C3 (precut E/P)
• R9 into pSB1C3 (precut E/P)
• R10 into pSB1C3 (precut E/P)

• transformation of 10 ul ligation reaction after heat inactivation into DH5alpha cells

14/09/2010

Gel 100914-1

Gel 100914-2

• Colony- PCR for the cloning products done on the previous day (gels 100914-1 and 100914-2 on the right)
• positive clones were Mini-Prepped and test digested

15/09/2010

Gel 100915-1

• PCR for the introduction of the Kozag-Sequence in front of the hRluc_BGH part; two strategies were performed in parallel using different touchdown-PCR protocols. Protocol nr. 1 was set up as follows:

• 25 ul Phusion HF MasterMix
• 0.5 ul of 100 um primers
• 1 ul of template R12 (50 ng/ul)
• 23 ul of water

Touchdown PCR was performed according to the following protocol:
................................................
95 °C/5 min
................................................ (1x)
95 °C/30 s
68 °C/45 s (- 0.5 °C/cycle)
72 °C/1 min
................................................ (35x)
72 °C/10 min
................................................ (1x)
4 °C/ forever
................................................

Protocol nr. 2 was set up as follows:

• 25 ul Phusion HF MasterMix
• 0.1 ul of 100 um primers
• 1 ul of template R12 (50 ng/ul)
• 23.8 ul of water

Touchdown PCR was performed according to the following protocol:
................................................
95 °C/5 min
................................................ (1x
95 °C/15 s
68 °C/45 s (- 0.5 °C/cycle)
72 °C/1 min
................................................ (15x)
95 °C/15 s
60 °C/30 s
72 °C/1 min
................................................ (20x)
72 °C/10 min
................................................ (1x)
4 °C/ forever
................................................

Each PCR was performed in two replicates. The result of the PCRs was analyzed on a 1 % agarose gel (100915-1), run for 1 h @ 100 V. The PCR nr. 2 was subsequently PCR purified by applying a Qiagen PCR purification KIT and used for the cloning

16/09/2010

• Cloning of Kozag_hRluc_BGH fragment into pSB1C3 and Constructs R24-R31
• Cloning of the XhoI/XmaI_suffix oligo via NheI/PstI into the TetR construct F16)

• Digestion of PCR fragment with SpeI/PstI according to 3A standard protocol; digestion of destination Vector pSB1C3
• Ligation and Transformation were done according to the standard protocol

17/09/2010

Gel 100917-1

Gel 100917-2

• Selection of colonies from the plates (see previous days' cloning) via colony-PCR using the standard sequencing primers; a ~5.5 kb fragment should be visible for the assembled construct K1-K8, if colony-PCR was positive (gel 100917-1 and 100917-2). No sample shows a 5.5 kb band, but that's most likely due to the very difficult amplification of such a long fragment with Fermentas 2x PCR MasterMix (Taq-based). Therefor 3 miniprep cultures were inocculated for each construct (K1-K8). The TetR oligo insertion and the Kozag_hRluc_BGH cloning into pSB1C3 gave the right bands on the gel.

19/09/2010

• Miniprep of the previous days inocculated miniprep cultures

20/09/2010

Gel 100920-1

Gel 100920-2

• test digestion of the miniprepped clones (previous day) with EcoRI/PstI; For all clones at least 2 digestions gave the exactly expected band (gel 100920-1 and 100920-2); therefor we can say, that the tuning construct is succesfully assembled and ready to be tested

21/09/2010

22/09/2010

• digestion of R23 with EcoRI and PstI in EcoRI Buffer (NEB) and ligation into digested Template:Part, transformation, growing on selective agar plates (chloramphenicol), following standard protocol recommendations

23/09/2010

Gel 100923-1. Analytical gel with following lanes: 1) till 8) colony PCR products with nice bands as expected at ~ 800 bp especially at lane 1, 4, 6 and 8, lane 9) 1kb Plus Ladder (Invitrogen)

• minipreps (using Quiagen Kit) and colony-PCR of each 8 colonies of R23 in Template:Part (digested, ligated, transformed and plated on previous day)
  • • PCR product of R23C on analytical gel (see gel 100923-1)
  • digestion of R23 and R33 with either SpeI and PstI or EcoRI and NheI
  • ligation into digested Template:Part, transformation, growing on selective agar plates (ampicillin), following standard protocol recommendations

24/09/2010

• "Something is rotten in the state of Genemark..." Lorenz 18:00, 24 September 2010 (UTC)

• inoculation and colony PCR of the transformed (23A+33A)=56C construct from yesterday. Same PCR protocol was used, even though the fragment should be 1100 and therefore elongation was probably not long enough. The PCR showed a band at 300bp, which dominik says is normal if you have religated vector. But nevertheless, miniprep of La1, La3 and La4 were made, because they didn't show a strong negative PCR band.
• test digestions (actually they are preparative, the only difference is I will test them on the gel as well to be sure I have the right thing)
  • R23A from yesterday (miniprep 8 and 8/) is digested with Eco&Nhe, expected fragment is 500bp
  • R33 with Spe&Pst, expected fragment 600bp
  • R56C with Spe&Pst, expected fragment 1100bp
  • R8 with Eco&Nhe, expected frament 1000bp
• ligation of R23A+R33+backbone C
• ligation of R56C + R8 + backbone A

25/09/2010

• PCR amplification of standardised pcDNA5 p55 and p1 with primer D47/48 and D55/69

26/09/2010

Gel 100926-1. Analytical gel with following lanes: 1) R33 E/N (from 23^rd September) 2) R23_1 S/P 3) R23_6 4) R33C 5) R23_4 6) R23_8 7) 1kb Plus Ladder (Invitrogen) 8) R33C E/N 9) R23_4 S/P 9) R23C_8 S/P

Gel 100926-2. PCR results of p55 and p1: p1 with primer 47/48 (lane 1), p1 with primer 55/70 (lane 2), p55 with primer 47/48 (lane3), p55 with primer 55/70

• miniprep of R23C cultures again (colonies from transformation (done on wednesday))
• check of all digestions on an analytical gel (see gel 100926-1)
  • fragment for R23_6 is a bit smaller than expected (rather 400 bp than 500 bp)
    • originally digested R23A runs slightly below 500 bp, too (data not shown)
      • because of reverse elements and resulting secondary structure?
  • repetition of ligation with R23_6 and old R33 (nice band at roughly 600bp)
    • including negative control at this time with only backbone Template:Part
  • transformation

tuning construct:

• gel extraction of PCR on p55 and p1 (see gel 100926-2)
• recloning of R1,R4 and R7 into standardbackbone Template:Part
  • digest 500ng of R1, R4 and R7 with EcoRI and PstI
  • heat inactivate 20 min at 80°C
  • Nucleotide removal
  • ligate 150ng and 250ng of insert into 30ng Template:Part 1h at RT and transform ligation

27/09/2010

Gel 100927-1: colony PCR on K1 (lane 1-8), K4 (lane 9-16) and K8 (lane 17-24)

Gel 100927-2: colony PCR K1 (lane 1&2), K4 (3&4) und K7 (5&6), K3 digested with BamHI (7), K3 digested NheI and SpeI (8), R33 in pSB1A3 digested with EcoRI and PstI (9), R33 digested with NheI and SpeI (10)

tuning construct:

• colony-PCR on K1, K4 and K7 cloned into Template:Part showed nearly only positives as a band of 400bp is expected by using the primer pair 50/75
• miniprep of K1, K4 and K7
• send for sequencing
• cloning of K3 into Template:Part:
  • digest K3 with PstI and EcoRI
  • ligate 200ng with 30ng of vector Template:Part
  • transform ligation and plate on an Ampicillin plate
• digest shRNA 6 (F11) and shRNA 7 (F12):
  • 1h at 37°C with HindIII
  • heat inactivate 20 min at 80°C
  • digest 1h at 37°C with AflII
  • purify by Nucleotide removal kit and elute in 20µl

repressor construct:

• a lot of religations, thus: a lot of colonies to pick
• colony-PCR and test digestion with SpeI and PstI of promising mini-preps revealed some positive samples
• ligation with R8 and R11, respectively (each digested with EcoRI and NheI) in Template:Part (backbone, digested with EcoRI and PstI), following standard protocol recommendations
• growth of transformed cells on selective agar plates (chloramphenicol) over night

28/09/2010

Gel 100928-1. Colony PCR of K3 construct

Gel 100928-2. Test digest of with PstI and EcoRI (lane 1-6), test digest with BamHI (lane 7-12), K1 (lane 1&2), K4 (lane 3&4), K7 (lane 5&6)

Gel 100928-3. Digestion of K1 (lane 1-4), K4 (lane 5-8) and K7 (lane 9-12) with HindIII and AflII

tuning construct:

• colony PCR of K3 (gel 100928-1)
• inoculation for mini prep of K3
• mini prep of K1, K4 and K7
• test digestion of K1, K4 and K7 with PstI & EcoRI and BamHI (gel 100928-2 and 100928-3)
• digestion of K1, K4 and K7 with HindIII and AflII prior to gel extraction
• ligation of shRNA 6 and 7 into K1, K4 and K7
• transformation of ligation
• plate ligation on ampicillin plates

repressor construct:

• re-do of mini-preps and digestions of (R23+R33)C_1, (R23+R33)C_14, (R23+R33)C_17 and (R23+R33)C_23 (promising)
  • check on gel, submission for sequencing next day
• analysis of transformations from yesterday (i. e. (R23+R33) + R8 or (R23+R33) + R11 in chloramphenicol standard backbone)
  • only re-ligations (as assumed from negative control), more colonies next day

29/09/2010

Gel 100929-1 first gel: double digestion of K1 and K3 with PstI and EcoRI: K1 (lane 1-4), K3 (lane 5-8), second gel: digestion of K1 and K3 with BamHI: K1 (lane 1-4), K3 (lane 5-8)

Gel 1000929-2 digestion of K1 and K3 with HindIII and AflII: K1 (lane 1&2), K3(lane 3-12)

gel 100929-3 pcr on binding sites for shRNA 10

tuning construct:

• mini Prep on K3 and K1 constructs
  • test digestion with PstI & EcoRI and BamHI showed all positive results
  • digest K3 and K1 with HindIII and AflII
• cloning of shRNA 6 and 7 into K1, K3, K4 and K7:
  • colony PCR on ligation from the 28/09/2010 did not reveal any positives by using the following protocol:
  • colony PCR on ligations from the 28/09/2010 did not reveal any positives by using the following protocol:
  • start from the beginning again: in doublicates:
  • first procedure:
    • digest PCR on shRNA 6 and 7 as well as K1, K3, K4 and K7 with HindIII and AflII
    • purify shRNAs with nucleotide removal kit
    • purify digested K3 and K1 with gel extraction kit
    • ligate K1, K3, K4 and K7 with shRNA 6 and 7
  • second procedure:
    • digest K4 and K1 as well as shRNA6 and 7 from PCR product and purify via nucleotide removal kit
    • ligate K4 and K3 with shRNA 6 and 7
  • transform all ligations
• cloning of binding sites:
  • second strand synthesis PCR on binding sites for shRNA 10

Gel 100929 r-4: colony PCR of repressor construct, band at 350bp points at backbone religation.

Gel 100929 r-5: test digestion of miniprep number 14 withe EcoRI and NheI shows expected band at 1100bp. Digestions of R8 and R11 were also positive, DNA cut out with SpeI and PstI runs at 800bp. Lane 1=miniprep 14 undigested, lane2=miniprep 14 digested, lane3=miniprep 23 digested, lane4=R8 undigested, lane5= R8 digested, lane6=R11 undigested, lane7=R11 digested

repressor construct:

• colony PCR after transformation of P56+R8 and P56+R11 (P56=R23A+R33) yielded no positive results
• miniprep of colonies 11, 14, 17 and 23 from P56 cloning were repeted because of bad miniprep results yesterday, then digested with S/P
• R8 and R11 were digested with E/N again
• digestion of P56 colony 14 was positive on the gel, digestion of R8 and R11 also gave the right fragments
• pBS 1C3 backbone was SAP treated to reduce religation that was likely to be the problem with previous transformations
• ligation of P56 (14) with R8 and R11 and SAPed backbone and control ligation with backbone only, transformation

30/09/2010

Gel 1000903-2 colony PCR on shRNA10 cloned into K3 and K4

Gel 1000930-3 colony PCR on shRNA10 into K3 and K4

tuning construct:

• colony pcr on shRNA 6 and 7 into construct K3 and K4
• cloning of binding sites for shRNA 10 into K3 and K4:
  • digestion of binding sites (perfect, imperfect 9-12 and imperfect 9-22) for shRNA10 with xhoI and ageI :) and construct K3 and K4 in psB1A3 with xmaI and xhoI
  • ligation of binding sites with digested constructs
  • transformation of ligation into TOP10 cells
  • plate on ampicillin plates ON

Gel 100930-1 colony PCR of Repressor construct with different promoters (R8 or R11). Positive colonies show an amplification product at 2500bp (lanes 1,2,4-8, 10, 13-16)

• plenty of colonies on the P56+R8 and P56+R11, NO colonies on control plate with SAPed backbone, colony PCR revealed 50% of positive clones

01/10/2010

Gel 101001-1. Digestion of miniprep no. 8, 9, 14 and 16 with EcoRI and PstI. Odd lanes are undigested controls, even lanes are digested minis

repressor construct:

• colony 8, 9, 14 and 16 were chosen for miniprep. They consist of the complete TetR construct in pBS 1C3
• digestion of the minis with EcoRI and PstI for cloning into Template:Part, SAP of Template:Part, digestion was tested on gel and proven positive (see Gel 101001-1)
• nucleotide removal, then ligation of TetR8 and TetR14 equimolar with SAPed backbone, TetR9 and TetR16 2:1 with not-SAPed backbone, transformation with TOP10 cells (10µl on 50µl E. coli)

Screening 1: shRNA10 single binding sites

Screening 2: shRNA10 single binding sites

• Screening of the binding sites cloned into the constructs K3 and K4 containing shRNA10 in pSB1A3 on the previous day. 16 colonies were picked for each perfect, rand9-12 and rand9-22 binding site and colony-PCR was performed according to the colony-PCR standard protocol (Primers Luc2_bs_screen_seq and sh10_ape_xma_screen_rc). The results are shown on the gels Screening1 and Screening 2. Indicated are the construct numbers (either K3 or K4, which differ in the promoter driving the Luc2 gene) and the binding site property (perfect, 9-12 randomized or 9-22 randomized). Almost all clones show a positive band, although only some have a really bright band at the size of ~ 250 bp. LB cultures for 10 clones for each constructs K3 and K4 were inocculated.

02/10/2010

Gel 101002-1. Colony PCR. All lanes with right amplified fragment except on lane 3, 10, 11, 15 and 23. Lane 1 & 14: 1kb Plus Ladder (Invitrogen)

Gel 101002-2. Test digestions of repressor construct. Upper pattern: EcoRI & PstI. Lower pattern: BamHI. All bands revealing positive results except in the first column (negative control from lane 3 of gel 101002-1). Lane 1 and Lane 10: 1kb Plus Ladder (Invitrogen). Lane 16, 17, 18: incomplete digestion

• Miniprep of the LB cultures (constructs K3 and K4 with shRNA10 and the corresponding binding sites) were done and used for transfection of Hek-293 cells

repressor construct:

• colony-PCR of 6 colonies for each plate of previous day transformations
  • analysis, see gel 101002-1
• twice test digestion of promising mini-preps, following standard protocol recommendations
  
  • first digestion with EcoRI and PstI to check for insert size
  • second digestion with BamHI to check the insert
    • see gel 101002-2: some positive results will be send for sequencing and typed T1 and T2, respectively
      • moreover: usage of one construct for transfection into HEK cells

03/10/2010

repressor construct:

• sequencing results were fine
• digestion and purification of T1 and T2 with XmaI and XhoI to clone in binding sites (against ON-targets) following standard protocol recommendations
• annealing of binding sites using oligos
  • D108 & D109: once binding site for miR122
  • D110 & D111: twice binding site for miR122 right behind each other
    • protocol:
      • 5 µl of each oligo, 5 µl of Buffer 2 (NEB), 35 µl H₂O
      • 95°C, 5'
      • cool down to room temperature for 2h
      • incubation on ice, 10'
      • use 1 µl for ligation with ~ 40 ng of backbone (i. e. digested T1 and T2)
• ligation (including to negative controls with backbone only to check for re-ligation without SAP treatment)
• transformation and growth on selective plates (ampicillin)

04/10/2010

repressor construct:

• colony-PCR of 6 colonies for each plate of previous day transformations
  • gel 101004-1 reveals almost positive samples
• submission of each a promising mini-prep for sequencing of binding site and correct insert

05/10/2010

repressor construct:

• sequencing results from previous day:
  • once or twice the binding site in each of the T1 constructs, respectively
  • once the binding site for the T2 construct
    • re-trial of ligation for two binding sites in T2
• planned transfection into HeLa and HUH cells (non-liver and liver to prove ON-targeting) in ratio 6:1 = repressor:operator(tuning) as recommended in the T-Rex manual

new approach:

• purification of PCR amplified shRNA-like miRNA against hAAT ("shhAAT")
• digestion of shhAAT and Q2 and Q3 with HindIII and AflII (for backbones sequential because of two inner AflII cutting sites)
• ligation in triplicates (using 70 ng of backbone and 10, 15 or 20 ng of insert, respectively)
• transformation, over-night growth on selective agar plates (ampicillin)

06/10/2010

• waiting for colonies from previous night, which did not grow after all

07/10/2010

• repetition of shhAAT cloning
• touch-down PCR setup: 8.5 µl ddH₂O, 0.5 µl template DNA (pcDNA5, 50 ng/µl), 0.5 µl each primer (i.e. D7 & D16, 10 µl Phusion PCR MasterMix (2x)
• touch-down PCR protocol: like this but with increase of annealing temperature from 68°C to 61°C (i. e. 0.2°C per cycle)
• go on like 05/10/2010 but growth on freshly prepared plates

08/10/2010

• mini-prep and submission for sequencing for the only promising colony of Q2 containing shhAAT for sequencing

09/10/2010

• sequencing yesterday was fine but construct contains TetO₂, thus: maybe Q2 and Q3 were confused
  • that would explain why measurements with TetR constructs are not working properly even though sequences were fine
• cloning of binding sites for shhAAT (perfect binding site generated via annealing, and randomized ones PCR amplified)
  • touch-down PCR setup: 17 µl ddH₂O, 4 µl each oligo (i.e. respective forward primer + second strand oligo D121), 25 µl Phusion PCR MasterMix (2x)
    • touch-down PCR protocol: like this but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) and four additional cycles with 62°C
  • annealing of perfect binding against shhAAT sites with an oligo-based strategy following the protocol from the 3^rd October
  • digestion of PCR amplified binding sites with AgeI and XhoI, digestion of Q2 backbone containing the shhAAT with XhoI and XmaI, afterwards heat inactivation, nucleotide removal and ligation following standard protocol recommendations
  • transformation and over-night growth on selective agar plates (ampicillin)

10/10/2010

Gel 101010-1. Colony PCR for construct containing shhAAT and binding sites. Expected is an amplified fragment a bit smaller than 300 bp which can be seen on lane 3, 4, 6, 7, 8 (perfect binding sites), 12, 13, 14, 16 and 17 (randomized binding sites). Lane 1 and Lane 26: 1kb Plus Ladder (Invitrogen)

• colony pcr reveals some positive clones (see gel 101010-1) from previous day
  • inoculation of 5 ml LB Amp cultures for mini-prep over night

11/10/2010

• mini-prep of ten cultures from yesterday and direct transfection to test the system
  • amount: each 100 µl (c=2.5 ng/µl)

measurement construct for single binding sites:

• PCR-based generation of binding sites against miR122 (perfect and randomized)
  • touch-down PCR setup:
    • 17 µl ddH₂O,
    • 4 µl each oligo (i.e. respective forward primer D71, D62 or D5 + second strand oligo D13),
    • 25 µl Phusion PCR MasterMix (2x)
  • touch-down PCR protocol:
    • like this but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x)
    • and four additional cycles with 62°C, elongation time only 10 seconds
• digestion of generated binding sites and the pSMB_miMeasure backbone with EcoRI and PstI, then nucleotide removal
• ligation with ~ 70 ng vector and between 7.5 ng and 15 ng insert
• transformation

12/10/2010

measurement construct for single binding sites:

• colony pcr of each eight of load of colonies + two negative controls (sum: 50 samples)
  • protocol: 16 cycles touch-down pcr from 70°C to 62°C of annealing temperature, then 19 additional cycles with 62°C annealing temperature
    • elongation time: 90''
  • no positive results, maybe due to insufficient screening primers (one for BBb standard, the other one for CMV resulting in an 1300 bp fragment)
    • as a consequence: repetition of entire cloning with SAP treated backbone tomorrow

13/10/2010

measurement construct for single binding sites:

• repetition of cloning from 11/10/2010
• note: vector concentration of pSMB_miMeasure is okay but after digestion and nucleotide removal it is always pretty low
  • (loss of 75% of DNA on average whereas other DNA yields to 90% of purified DNA as compared to digested amount)

14/10/2010

• cloning of 47 - 58 into pSB_H1
• cloning of tuning constructs for in vivo measurement:
  • cloning of binding sites of miRNA haat with perfect and several imperfect binding
  • oligo annealing following the standard protocol
  • digestion of backbone pbsU6 with NotI and XhoI
  • ligation and transformation

15/10/2010

Screening of M1 to M11 constructs, 4 clones were picked from each plate. 300bp band marks positive clones

Screening of M12 to M22 constructs, 4 clones were picked from each plate. 300bp band marks positive clones

Screening of measurement standard with perfect, 9-12 and 9-22 randomized binding sites. constructs, 8 clones were picked from each plate. 300bp band marks positive clones

• screening of plates M1 to M11 of the transformation with the construct pBSU6_SV40_Luc2 with 11 different binding sites for sh hAAT. Four clones from each plate were screened by colony pcr, positives give a band at 300bp. Primers Luc2_bs_screen_seq_fw and reverse primerse are the reverse binding site oligos for the binding sites.
  • inoculation of miniprep cultures for one positive from each plate.

• transformation of miRsAg and mir122 expression constructs

• screening of measurement standart cloning from 13th with perfect, 9-12 randomized and 9-22 randomized binding sites for miRNA 122. primers mir122_mimeasure_screen_rev and pSMB_miMeasure_screen_fw. Positives give a band at 300bp.
  • inoculation of miniprep cultures for positive clones

• screening of plates M12 to M22 of the transformation with the measurement plasmid with 11 different binding sites for miRsAg. Four clones from each plate were screened by colony pcr, positives give a band at 300bp. Primers pSMB_miMeasure_Sequencing_rev and forward primerse are the forward primer oligos for the binding sites.
  • inoculation of miniprep cultures for one positive from each plate.

16/10/2010

• mini Prep of mimeasure colonies (4 each)
• mini prep of haat constructs
• inoculation of miRsAg and mir122 expression constructs
• Maxi Preps of constructs M1 till M10
• inoculation for Maxipreps of Adenovirus 5, AAV8 rep and cap genes and pBSU6 H1 haat construct
• inoculation for Maxiprep of constructs M11 an M23-29
• cloning of on targeting constructs with a new strategy...

17/10/2010

• annealing of eleven sAg binding sites using oligos
  • • protocol:
      • 5 µl of each oligo, 5 µl of Buffer 2 (NEB), 35 µl H₂O
      • 95°C, 5'
      • cool down to room temperature for 2h
      • incubation on ice, 10'
      • use 1 µl for ligation with ~ 40 ng of backbone (i. e. with NotI and XhoI digested pBS_U6 containing hAAt cDNA)
• transformation and growth on selective plates (ampicillin)

18/10/2010

• colony-PCR of 4 colonies for each plate of previous day transformations using reverse binding site oligo and forward hAAT primer expecting a fragment round about 1300 bp
  • gel 101018-1 reveals almost positive samples except two (data not yet shown)

19/10/2010

• mini-preps of cloned shAAT constructs
• dilution and co-transfection with miRsAg expressing construct into HeLa cells for ELISA measurements