Team:TUDelft/Notebook/RNAi
Notebook
RNAi
Week 24
Week 25
Week 26
Week 27
Week 28
Week 29
Week 30
Week 31
Week 32
Week 33
Week 34
Week 35
Week 36
2/9
Alicia
Alicia: Plating of plasmids pBbA2k-GFP, pBbE-GFP and pBbB7a-GFP. The plasmids pBbA2k-GFP, pBbE-GFP and pBbB7a-GFP were plated in LB agar plates containing kanamycin, chloramphenicol and ampicillin respectively.
Week 37
7/9
Javier
BglBricks backbones: Primers pGibs-B7a Fw and Rv, pGibs-A2k Fw and Rv, and pGibs-E8c Fw and Rv were resuspended [Primer working stock] to reach a concentration of 100uM.
Alicia
Cultures of plasmids pBbA2k-GFP, pBbE-GFP and pBbB7a-GFP for Mini-prep: Cultures were prepared from the LB agar plates (04-09-2020), consisting on 10 ml of LB media, 10 µl of antibiotic and a single colony from a LB agar plate. The corresponding antibiotics were kanamycin for pBbA2k-RFP, chloramphenicol for pBbE8c-GFP and ampicillin for pBbB7a-GFP. The cultures were grown overnight at 37ºC, 200 rpm.
8/9
Javier
Mini-preps were done of colonies cultured with [PureYield Plasmid Miniprep System.] The results were the following: B7a (107.5 ng/ul) A2k (64.8 ng/ul) and E8c (154.7 ng/ul).
Plasmid were diluted to 10ng/ul (0.93ul of B7a, 1.543ul of A2k, 0.646ul of E8c; diluted with NF water to 10ul).
PCR was performed with plasmids B7a A2k and E8C from minipreps as templates with Gibson assembly primers, in order to amplify the backbones with primers pGibs-B7a Fw and Rv, pGibs-A2k Fw and Rv, and pGibs-E8c Fw and Rv, respectively. Q5 was the polymerase used because of its high fidelity [Q5 PCR].
9/9
Iris
An agarose gel was run with 5ul of PCR products to check the sizes of the amplicons. [AGAROSe GEL] On the gel, amplicons showed the correct size.
As the bands were sharp and no by-products were seen, PCR products not loaded were purified using PCR Cleanup kit [PCR CLEANUP PROMEGA]. Nanodrop values showed a good concentration of the backbones: B7a: 92,5 ng/ul, A2k: 109 ng/ul, E8c: 100 ng/ul.
PCR purification of the remaining PCR product (~45µL). Followed the Wizard SV Gel and PCR Clean-up system. Last step adjustment to protocol: added 40µL of warm nuclease free water.
Nanodrop to measure backbone concentrations. Gibson Assembly of B7 backbone + insert (Fox1, Fox1*, DRDB). Followed the protocol of [NEBuilder HiFI DNA Assembly Cloning Kit.] Transformation of newly made plasmids according to the protocol of the kit: [NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol]. Plated positive, negative, Fox1, Fox1* and DRDB on different Ampicillin plates.
Javier
shRNA: Template primers annealed (primers T7_shRNA, T7_shRNA_RC, T7_shRNA* and T7_shRNA_RC) for transcription [Protocol]. shRNA were produced at 42ºC using RiboMax kit [RIBOMAX PRODUCTION]. The product of the reaction was stored at -20ºC.
10/9
Iris
Plate results: colonies were grown in all the plates except the negative controls.
Picked 3 colonies from each plate (Fox1, Fox1*, DRDB), grew overnight in 10mL agar, 10µL Ampicillin.
Javier
shRNA:shRNA produced were DNAse treated [DNASE TREATMENT RIBOMAX] and RNA was purified with minElute columns [QUIAGEN MINELUTE]. High yields of shRNA and shRNA* were obtained, as measured by the nanodrop:
- shRNA: 3.258 ng/ul, A260/A280 = 2,11, A260/A230 = 2,32
- shRNA*: 3.158 ng/ul, A260/A280 = 1,98, A260/A230 = 2.26
11/9
Iris
Mini-prep of the overnight cultures with [PureYield Plasmid Miniprep System]. Did 6 mL of each sample. Results of the Nanodrop: ~ 80 ng/µL
Prepared sequencing mixture: 8µL plasmid, 2µL primers (10µM).
Javier
An Urea-PAGE gel was run [DENATURING UREA PAGE] with 1ul of purified shRNAs and 2ul of ladders. Bands from the shRNAs could be seen, but the sharpness of the bands and the quality of the resolution were low. Fixation en methanol/Ethanol 10% for 5min, 12% 19:1, constant 25mA for 2h.
Week 38
14/9
Iris
Sent the 9 tubes to Macrogen Europe for sequencing
Javier
Urea PAGE gel was repeated [DENATURING UREA PAGE], following a fixation in metanol/ethanol 10% for 5min, 12% 19:1, constant 15mA for 2h. 1:5 dilution was done with the shRNA batch at 42ºC. 1 ul of each dilution were loaded in the gel, although nothing but the ladders could be seen.
16/9
Javier
shRNA: Urea page gel was performed again [DENATURING UREA PAGE], with 12% acrylamide ratio 19:1, fixation was done in met/et for 30sec, constant 120V ~2h.
1ul direct from stock solution of produced RNA was loaded in each lane. Bands could be seen clearer. Expected sizes were higher (65bp) than the ones seen in the gel: this could have been due to a strong secondary structure formation.
The sizes of the hairpins appear to be about 30bp in stead of 65bp, but hairpins had been reported to move faster in the gel because their secondarys structure does not denaturalize even in UREA, as was read in literature data.
17/9
Iris
Received sequencing results. Javi checked it; F and M were fine. F1 and M2 will be used for further experiments. DRDB went wrong;
18/9
Iris
Transformation of F1 and M2 in 50 µL BL21; Used NEB Transformation Protocol for BL21(DE3) Competent Cells (C2527). Added 1µL of F1 or M2 plasmid.
Plated transformed cells (50µL) on ampicillin plates, left them at RT over the weekend.
Week 39
21/9
Gabriela
Cells grew on the plates. Per plate, Fox and Fox*, one colony was picked; from each colony 2 pre-cultures were grown (10mL, 10µL Amp) overnight at 37 degrees. In addition a culture of BL21 was grown as a control.
Javier
shRNA: Two new batches of shRNAs were produced at different temperatures (30 and 37ºC) to see how temperature affects hairpin production. [RIBOMAX PRODUCTION].. The product was purified using columns [QUIAGEN MINELUTE]., without prior DNAse treatment.
nanodrop: All products yielded products, except for negative controls.
An Urea Gel was run with 1:0, 1:5, 1:10, 1:20 dilutions of the 42ºC produced batch to elucidate which concentration is appropriate for band visualisation in [EMSA] assays. 1ul of each dilution were loaded along with all samples from 37ºC and 30ºC batches produced.
22/9
Iris
Cells were diluted to 0.05 OD in a total volume of 25 mL (+ 25µL Amp for Fox and Fox*).
Cells were grown to OD 0.6. Then 2 samples for each were taken (1.5 mL each) and centrifuged. Pellet was stored at -20 degrees. IPTG was added to all samples (1mM (150µL [100mM] IPTG to 15mL cells). 4 hours of incubation at 37 degrees. Then 2 samples each were taken (1.5 mL; all pelleted) and remaining culture was stored overnight at 30 degrees.
Cells were grown to OD 0.6. Then 2 samples for each were taken (1.5 mL each) and centrifuged. Pellet was stored at -20 degrees. IPTG was added to all samples (1mM (150µL [100mM] IPTG to 15mL cells). 4 hours of incubation at 37 degrees. Then 2 samples each were taken (1.5 mL; all pelleted) and remaining culture was stored overnight at 30 degrees.
Second pre-culture was prepared in a glycerol stock (protocol Gabs email) and stored at -80 degrees in the freezer.
| t | 0h | 4h (1/4) | O/N (1/8 |
|---|---|---|---|
| BL21: 0.06 | 0.62 | 0.8 | 0.60 |
| FOX: 0.06 | 0.64 | 0.43 | 0.43 |
| Fox*:0.06 | 0.60 | 0.43 | 0.45 |
Javier
B. subtilis strain 168 was provided by Martin from an o/n culture. Glycerinates were done and stored at -80ºC [Glycerinates].
Genome was extracted from B. subtilis 168 and E. coli BL21 using [GENOME EXTRACTION GRAM +]. Although E.coli is gram negative, the protocols between these two types of bacteria differ by longer incubation times and higher lysozymes concentrations for Gram+, so extraction could be performed using Gram+ protocol for both types. High yields of purification: B. subtilis (198 ng/ul), and E. coli (309 ng/ul).
A PCR was performed with the extracted genomes in order to amplify YmdB and mini-3 from E. coli and B. subtilis, respectively. Primers used were YmdB_Fw & Rv, and mini3_Fw & Rv, and Q5 polymerase to ensure high fidelity [Q5 PCR].
Agarose gel was made to check the sizes of the amplicons [AGAROSE GEL] and the bands were gel purified to eliminate Genomic DNA contamination [GEL PURIFICATION].
- YmdB: 105 ng/ul, A260/A280 = 1'84, A260/A230 = 1'5.
- mini3: 104 ng/ul, A260/A280 = 1'84, A260/A230 = 2'11.
lhRNA: A PCR of fragments A ad B was performed using Q5 polymerase [Q5 PCR]. eGFP plasmid was used as a template and primers lhRNA_FragA_Fw & Rv, and lhRNA_FragB_Fw & Rv.
An agarose gel was made to check the sizes: Frag.A = 729bp, Frag.B = 717bp.
PCR product was purified [PCR CLEANUP PROMEGA], not from the gel but directly from PCR tube to avoid mutations due to UV light.
- lhRNA_FragA = 206 ng/ul, A260/A280 = 1'86, A260/A230 = 2'12.
- lhRNA_FragB = 179 ng/ul, A260/A280 = 1'85, A260/A230 = 1'93.
23/9
Javier
lhRNA: A Golden Gate assembly was performed [GG PROTOCOL] in a 25ul tube with 4ul of T4 ligase, 2'5ul of BsaI-HFv2, and 8ul of each of the fragments stocks, to ligate both fragment A and Fragment B from the lhRNA. Higher concentrations of enzyme and ligase were used to ensure the ligation of the high concentrations of DNA used.
Iris
Remaining culture was pelleted. 3 mL cells per eppendorf tube. Stored in the 4 C fridge.
Samples (1x each; BL21, Fox, Fox* of 0t, 4t, O/N) were resuspended in 100µL NF water. Then 10µL of each was combined with 10µL 2X Laemmli Sample Buffer. Then boiled for 10min at 95 degrees. Spin down + load on SDS-PAGE. Gel was washed with water, and then stained overnight with Coommassie Brilliant Blue.
24/9
Javier
Gel was destained (water + tissue) and a picture was taken on the Geldocs.
From the gel it was seen that the 4h of induction + O/N samples contained our proteins. The control (BL21) did not contain a band at this size. We chose to purify the proteins from the O/N sample as this sample contained the most proteins (thickest band).
Two pellets of each were combined in a total volume of 700µL NF water. These were resuspended. The 2 samples, Fox and Fox*, containing 700µL NF water + resuspended pellet of 6mL cells, were used for the His purification kit.
Protein concentrations were measured.
The flow through, wash 1, wash 2 and the purified protein were all stored. 10µL of each sample was combined with 10µL 2X Laemmli Sample Buffer. Then boiled for 10min at 95 degrees. Spin down + load on SDS-PAGE. Gel was washed with water, and then stained overnight with Coommassie Brilliant Blue
Maartje
To obtain a plasmid carrying sgRNA targeting the BL21 RNC, two PCRs were performed on pKDsgRNA-p15. The forward primer RncF and reverse primer BetaR were used for the first reaction, and the forward primer PkdseqF with the reverse primer RncR for the second reaction. The protocol for Q5 polymerase was followed. The PCR products were checked on a 1% agarosegel and subsequently a Gibson Assembly was done with the two obtained PCR fragments. The products were transformed following the protocol for heat shock transformation into dh5alpha. The cells were recovered at 30C and plated on LB-spec.
To obtain the YMDB plasmid with the A2k backbone and Mini3 on E8c, a Gibson Assembly was performed with these two products. Both products were transformed following the protocol for heat shock transformation into dh5alpha. The cells were recovered at 37C and plated on LB-kan for YMDB and LB-Cam.
Javier
An agarose gel was made to check the ligation of the lhRNA insert from yesterday.
Ligation seemed to be very inneficcient. The band corresponding to the correct size was excised and gel purified [GEL PURIFICATION].. Only 6 ng/ul were able to be purified, which was not enough for downstream applications. Ligation has to be performed with traditional restriction-ligation protocols.
25/9
Javier
Gel was destained (water + tissue) and a picture was taken on the Geldocs:
lhRNA: A PCR using Q5 [Q5 PCR] was performed again with eGFP plasmid as the template and primers lhRNA_FragA_Fw & Rv, and lhRNA_FragB_Fw & Rv. 3 PCRs for each fragments were performed to ensure enough DNA was produced for downstream use.
An agarose gel was prepared to check the sizes of the amplicons [AGAROSE GEL] . Correct sizes of the bands could be seen, except fot the 3rd PCR of fragment A.
PCR cleanup was used [PCR CLEANUP PROMEGA] directly from the PCR products, with 6ul of NF water as elution, to concentrate the DNA at its maximum possible.
BsaI was used for the digestion of the lhRNA purified. [RESTRICTION PROTOCOL]. 3ul of BsaI-HFv2 with 8ul of each lhRNA fragments in a 50ul reaction tube.
BsaI was used for the digestion of the lhRNA purified. [RESTRICTION PROTOCOL]. 3ul of BsaI-HFv2 with 8ul of each lhRNA fragments in a 50ul reaction tube.
A ligation using T4 ligase [T4 LIGASE PROTOCOL] was used to ligate fragment A and B together. 5ul of T4 ligase was added to 10ul of the purified PCR cleanup product in a 20ul reaction.
An agarose gel was made to check for the ligation product, where all the 20ul reaction were loaded in the gel to purify the band needed.
Purification from the gel was made to recover only the correct ligated fragment. [GEL CLEAUP PROTOCOL]. Purified product: lhRNA_insert, 33 ng/ul.
27/9
Javier
lhRNA: lhRNA purified insert was cloned into pBbB7a_backbone using the Gibson Assembly protocol [NEB HIFI ASSEMBLY]. Transformation was done by electroporation in electrocompetent E.coli BL21 cells. [ELECTROPORATION].
In order to generate a higher stock of lhRNA_insert, a PCR using lhRNA_fragA_Fw and lhRNA_fragB_Fw primers were used to PCR amplify the insert generated by ligation on ht e25-09-2020. Two polymerases (Q5 "GC enhanced", and TAQ) were tested to elucidate which can polymerise from a template with a difficult and strong secondary structure. [Q5 PCR GC enhanced, TAQ PCR]. This information was useful to assess whether lhRNA transcript could be qunatified by qPCR, a reaction dependent of a sucessful polymerisation.
The PCR products were checked in an agarose gel to check amplicon sizes [AGAROSE GEL]: None of the polymerases were able to successfully amplify the insert. A long smear of higher molecular weight than individual fragments could be seen suggesting inter-molecular hybridisation of intermediate products. TAQ could amplify one part of the template, terminating the transcription somewhere near the loop region, as could be judged by the size of the amplicon.
The primers used had a common ~22nt region that hybridises with the extremes of the hairpin. A new set of primers hybridysing more specifically to only one of the extremes out of the hairpin region were designed and commanded.
Week 40
28/9
Maartje
Mini3 and lhRNA with B7A backbone were transformed into BL21(DE3) electrocompetent cells following the protocol for [electroporation] and recovered at 37C and plated on LB-Amp.
Javier
shRNA: Another batch of shRNA at 42ºC was produced for later experiments [RIBOMAX PRODUCTION]., and was stored at -20ºC before DNAse treatment and purification. Buffers were made for DICER and Binding assays. Colonies of YmdB A2k and mini3 E8c were picked and grown overnight in respectively LB-Kan and LB-Cam.
29/9
Gabriela
YMDB A2k and Mini3 E8c constructs were isolated following the protocol for [PureYield Plasmid Miniprep System.]
Javier
EMSA: Shift assay was performed with 20 fmol of shRNA in presence of different ratios of Fox-1 RBD, in an 8ul reaction. [EMSA loading calculations]. Dilution of 1:5 was not sucessful, or Fox was not loaded. The interaction seems strong, as 1:1 ratio seems enough to produce a shift in mobility.
30/9
Javi
DICER assay: 1 mM concentration of ATP was added into the Dicer buffer 2x.
1 mM concentration of ATP was added into the Dicer buffer 2x.
shRNA and shRNA* were processed in Dicer buffers with and without ATP. It seems that ATP is not needed for the reaction, as the siRNA band produced from shRNAs is the same intensity in all cases. Dicer concentration: 0.25 uM, shRNA: 400ng in a 4ul reaction. Conditions: 1h incubation at 37ºC.
1/10
Javi
Dicer assay: Incubation was now performed at different time points. shRNA was the only one tested, as yesterday's results indicated that both seemed to be processed equally. Dicer concentration: 0.2 uM, shRNA: 20fmol (400ng) in a 10ul volume. Dicer seemed to process the shRNA, reachig its maximal total production at around 2h incubation.
2/10
Javi
lhRNA: New primers arrived: lhRNA-insert-Fw & Rv, lhRNA-T7-Fw. PCR was done using Q5, KOD and Taq. [PCR Q5, KOD and TAQ]. An agarose gel was run to see amplicon sizes, but nothing could be seen but the ladders: there was no amplification. Hairpins seem to be difficult structures to amplify, and qPCR might not be feasible.
Week 41
Week 42