Transformation
1, start with 5ul plasmid / product
2, mix it with 50ul bacteria (-80 freezer in cell culture room)
(if start with finished plasmid, then 5ul aliquots should be fine)
3, Put the mixture on ice for 30 min
4, Heat shock mixture for 45 seconds on 42 degree temperature
5, Put back it to ice for 5 min
.//6, Proliferate the bateria in 500ul Soc medium (super enriched LB) for 30 mins in shaker.
(can find it in -80 freezer in cell culture room)
7, Discard the majority of supernatant and resuspend the sediment with 100ul or so LB.
8, Distrubute it on plate with corresponding anti-botic property.
Gel preparation
Purfication the fregment from PCR or digestion of backbone
1, Two steps, gel migration at 1% and gel purification kit.
RNA extration and library preparation
Some tips for RNA extration
1) Performing multiple RPE wash steps to remove residual salts.
2) Perform slightly longer and slightly faster centrifugation steps.
3) Ensure that the procedure is performed at room temperature (please do not place samples on ice or spin at 4 deg C).
4) Ensure that the sample is not too cold prior to addition of Buffer RLT.
5) Ensure that the RLT buffer does not contain any precipitates. If there are precipitates, equilibrate the buffers at room temperature prior to use.
- Make sure Buffer RLT and centrifugation steps are done at room temp (it may crystalize if too cold)
- When washing, roll the column around to make sure the entire inside gets washed
from paper of Tissue wide PRC2 during corticogenesis.
RNA extraction and complementary DNA library preparation of MADM samples for RNA-seq
After cell sorting of E13/E16 and P0 cells, samples were incubated for 30 min at 37°C and stored at −80°C until further usage. After thawing samples on ice, their total volume was adjusted to 250 μl using RNase-free H2O (Thermo Fisher Scientific), followed by the addition of 750 μl of TRIzol LS (Thermo Fisher Scientific) and inverting five times. After incubating for 5 min at RT, the entire solution was transferred into a MaXtract tube (QIAGEN). Chloroform (200 μl) (Sigma-Aldrich) was added. After three times 5-s vortexing and 2-min incubation at RT, samples were centrifuged for 2 min with 12,000 rpm at 18°C. Supernatant was transferred to a new tube, and isopropanol (Sigma-Aldrich) was added in a 1:1 ratio. For better visibility of the RNA pellet, 1 μl of GlycoBlue (Thermo Fisher Scientific) was added and the entire solution was mixed by vortexing 3 × 5 s. Samples were incubated at −20°C for three nights to precipitate RNA. Then, samples were centrifuged for 20 min with 14,000 rpm at 4°C. Supernatant was removed, and RNA pellet was washed with 70% ethanol, followed by a 5-min centrifugation step (14,000 rpm at 4°C). RNA pellet was resuspended in 12.5 μl of RNase-free H2O. RNA quality was analyzed using a Bioanalyzer RNA 6000 Pico kit (Agilent) following the manufacturer’s instructions. RNA samples were pipetted into a 96-well plate, sealed with aluminum seal, and stored at −80°C until further use. Sequencing libraries were prepared following the Smart-Seq v2 protocol (64) using custom reagents (VBCF GmbH), and libraries from a 96-well plate were pooled, diluted, and sequenced on HiSeq 2500 (Illumina) using v4 chemistry or NextSeq550 (Illumina).
