Custom Hairpins

Customized pGIPZ vector creation

Updated 11/15/2010 dw

Ordering the 97mer
Check on UCSC genome browser to see 4 things:
 * Is your transcript annotated, and are there GIPZ hairpins already available on Open Biosystems (search transcript ID)
 * Is your transcript of interest expressed in your experimental cell type? you can use RNA seq data for this if applicable (Caltech RNA)
 * Are there multiple isoforms? (If there are, make sure you are hitting all of them)
 * Is there an affymetrix probe to your gene/lincRNA? If so, you can mine more information from it, potentially.

Get the mRNA sequence of your transcript. Important: you are going to input the TARGET sequence, which means it needs to RNA, not the DNA that it is coming from. You can get the RNA sequence from 2 places:

If your RNA is annotated, click on it and grab the mRNA sequence.

Otherwise, hover over the common region on UCSC genome browser, go to DNA (tab on the top). Make sure that you are getting the RNA molecule To this end, pay attn to strandedness. If transcribed from left to right, the DNA that comes out will be right. Otherwise, if transcribed right to left, get reverse complement of what comes out.

Go to the following website: http://cancan.cshl.edu/RNAi_central/RNAi.cgi?type=shRNA. Select shRNA_psm2, sequence info, human, Don’t check resolver. Click next

Check the box denoting that you have sequence (note that if your transcript has a refseq accession, you could use that). Click ‘retrieve oligo’

What comes out will be your 97mers, with the pink as the variable region corresponding to your RNA, the middle is the loop, and the sides have the mir-30 flanking sequence to allow it to work in the GIPZ vector. You can visualize where it is, by clicking ‘visualize’ (see screen shot).

You can load these sequences directly into an excel spreadsheet to facilitate ordering from the PAN facility on campus (you could also go through Elim, but it would be >10x more expensive and more slowly).

Cutting pGIPZ empty backbone
After prepping up pGIPZ  EMPTY  backbone (not scrambled), cut 3-6 ug of DNA for 2 hours with the following reaction for a 30ul reaction:
 * DNA
 * H20
 * 3ul NEB4
 * 3ul 10x BSA (1:10 dilution of NEB BSA)
 * 1.5ul BamHI-HF (NEB)
 * 1.5ul XhoI (NEB)

This next step is controversial. You should be able to gel-purify this reaction once cut and have your backbone ready to go. However, for unknown reasons, there have been problems with gel purifying this in the past. To get around this, you can just PCR purify this restriction digest (because you are just opening up the vector and no length of the backbone that would survive column purification would make it) and use that, keeping in mind that you will have to screen your colonies for empty vector contaminants.

Spec your cut vector.

PCR’ing restriction site-containing ends onto the 97mer
This protocol will assume that you are getting your 97ers from the PAN facility on campus and that the concentration of those oligos is 20uM in lyophilized form. You need to dilute this stock 10,000 fold. So add 100ul of water to each tube, vortex briefly (there is a resin deposit at the bottom of the tube, don’t try to resuspend this, but if you do, no worries).

Take 1ul of this master stock and dilute it in 99ul of water. Add 1ul of this dilute 97mer template to a PCR tube.

Assemble the following master mix on ice (per reaction)
 * Water 32.5ul
 * ExTaq Buffer 5.0ul
 * dNTP 4.0ul
 * DMSO 2.5ul
 * XhoI universal primer (10uM) 2.5ul
 * BamHI universal primer (10uM) 2.5ul
 * ExTaQ Enzyme 0.25ul

Add 49ul of this master mix to each tube containing 1ul of your 97mer template. Mix then put back on ice.

Use the following as your PCR protocol
 * 94°C for 1 min
 * 30 CYCLES
 * 94°C for 30 sec
 * 60°C for 30 sec
 * 72°C for 1 min
 * 72°C for 2 min
 * 4°C forever

Take 5ul of the PCR reaction and run it out on a gel to check your amplicon. It should be just over ~130bp.

Run the remaining 45ul of the PCR reaction through a PCR cleanup (Qiagen), and elute in 30ul of EB at the end (rather than the normal 50ul).

Cut this cleaned reaction with the following setup per reaction
 * 30ul of cleaned PCR amplicons
 * 4ul NEB4
 * 4ul 10x BSA
 * 1.5ul BamHI-HF (NEB)
 * 1.5ul XhoI (NEB)

Cut for 1.5 – 2 hours at 37°C, then PCR cleanup this reaction and elute in 50ul

Ligate ~50ng of cut backbone with ~1ml to 0.1ml of your insert. The ligation should be a 20ul reaction with NEB T4 ligase or NEB quick ligase. Do 1 hour for normal T4 ligase at room temp. ADD TO THAT

Transform 5ml of this reaction into Max. Efficiency STBL2 bugs (also flush this out, store at 4°C, etc.) Heat shock 42°C for 30 seconds, back to 4°C for 2 minutes. Directly plate onto WARMED LB amp. and incubate overnight (NOTE: STBL2 cells grow much less efficiently, so you may have to wait longer)