- Aspirate media, wash plates with PBS 1X and aspirate off excess liquid
- Add 0.5 ml Trizol to each well in the hood, pipet up and down a few times to cover the wells, pipet the mixture into autoclaved microfuge tubes, incubate for 5’ at RT (combine the replicates into one tube)
- Add 0.2 ml Chloroform to each tube, cap tightly and shake vigorously for 15 seconds, incubate at RT for 2-3 min.
- Spin at 12,000 rpm for 15 minutes at 4C, RNA will be in the aqueous top phase.
- Transfer the aqueous phase to a fresh microfuge tube
- Add 500 ml of RNA only isopropanol to each tube, incubate at RT 10’
- Spin at 12,000 rpm for 10 minutes at 4C, look for RNA pellet
- Wash the pellet once with 1ml 75% ethanol prepared with Rnase-free water, vortex the sample to mix and spin at 7500 rpm for 5 min
- Remove the ethanol, invert tube and allow to air dry for 10-15 min
- Resuspend the pellet in 50 ml of Rnase-free water, incubate for 10’ at 50C to dissolve, store at –80
SOFT AGAR ASSAY FOR COLONY FORMATION
Note: All volumes are calculated to cater for four plates per point. Values in bold represent plating in 6cm dishes.
Base Agar
1. Melt 1% Agar (DNA grade) in microwave, cool to 40°C in a waterbath. Warm 2X RPMI + 20% FCS to 40°C in waterbath. Allow at least 30 minutes for temperature to equilibrate.
2. Mix equal volumes of the two solutions to give 0.5% Agar + 1X RPMI + 10% FCS.
3. Add 1.5mL/ 35 mm dish (2.5mL), allow to set. The plates can be stored at 4°C for up to 1 week.
Top Agar
1. Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40°C in a waterbath. (It is important not to exceed 40°C, otherwise cells will be killed). Also warm 2X RPMI + 20% FCS to the same temperature.
2. Trypsinise cells and count. It is very important to have a positive control line (eg. ras transformed).
3. You require 5,000 cells/35mm plate (10,000/6cm plate), therefore you need 20,000 (40,000)/tube for four plates. Adjust cell count to 200,000 (400,000) cells /mL.
4. Add 0.1ml of cell suspension to 10ml yellow capped centrifuge tubes.
5. Label 35mm petri dishes with base agar appropriately (it is a good idea to remove the plates from 4°C about 30 minutes prior to plating to allow them to warm up to room temperature).
6. For plating add 3mL (5mL) 2X RPMI + 10% or 20% FCS and 3mL (5mL) 0.7% Agar to tube with cells, mix gently and add 1.5mL (2.5mL) to each replicate plate (usually plate out in triplicate). NOTE: Only do one tube at a time so that agar does not set prematurely.
7. Incubate assay at 37°C in humidified incubator for 10 – 14 days.
8. Stain plates with 0.5mL of 0.005% Crystal Violet for >1 hour, count colonies using a dissecting microscope.
Method 2.
Solutions
3.3% agar | autoclaved
1.8% agar |
2x DMEM
lx DMEM
FBS
1. Set up water baths to 37°C and 45°C
2. Prewarm 2x DMEM, DMEM and FBS to 37°C
3. Boil 3.3% agar to dissolve. Allow to cool, then place all media and Agar in 45°C water bath for 10 min.
4. In 50 ml tube mix:
3 mL 3.3% agar
5 mL 2x DMEM
10 mL 1x DMEM
2 mL FBS
5. Pipet 2 ml/well into 6 well clusters
6. Allow to set without disturbing in hood for 30 min.
7. Turn water bath to 41°C
8. Boil 1.8% agar to dissolve; allow to cool
9. Transfer 2x DMEM, FBS and agar to 41°C water bath for 10 min.
10. Detach cells and dilute to 3.4 x 105 cells/mL in DMEM
11. In 50 ml tube mix:
3 mL 1.8% agar
6.7 mL 2x DMEM
3.6 mL DMEM
1.7 mL FBS
12. Allow to cool for 20 min then add 2 mL cells
13. Carefully pipet 0.5 mL on top of base layer of agar and allow to set in hood for 30 min. Then transfer to incubator.
Preparation of RNA from tissues with Trizol (PJ Keller)
RNA from Tissue with Trizol (Click to Download)
*Keep tissue on dry ice until homogenization
- Add trizol to tissue, homogenize with mortar and pestle, pass through a 20G needle 2-3X to shear genomic DNA
- Incubate for 5 min at RT, keep on ice until all samples are homogenized
- Add 0.2 ml chloroform per ml Trizol, shake 15 sec, incubate 2-3 min at RT
- Spin at 12,000 x g for 10’ at 2-8C
- Transfer the clear aqueous phase to a new tube, add 0.5 ml isopropyl alcohol
- Incubate at RT 10’, centrifuge at 12,000 x g for 10’ at 2-8C
- Remove the supernatant, wash the pellet with 1 ml 75% EtOH, vortex
- Spin down pellet at 7500 x g for 5’ at 2-8C
- Air dry the RNA pellet 5-10’, resuspend in 110 ml RNase-free H20, remove 10 ml as an aliquot for pre-cleanup analysis
Use Qiagen RNeasy kit to clean up RNA prep (note-may want to add on-column DNAase digestion, see protocol with kit) - Make up buffer RLT (3.5 ml) by adding 35 ml BME to 3.5 ml RLT
- Add 350 ml RLT to 100 ml Trizol RNA sample, mix
- Add 250 ml 100% EtOH to the diluted RNA, mix
- Add the sample to a mini column placed in a 2 ml collection tube
- Spin 15s at 10,000 rpm
- Transfer the column to a new collection tube, add 500 ml RPE, spin 15s
- Discard the flow through, wash again with 500 ml RPE, spin 2’
- Discard Flow through, spin again 1’
- Elute into a 1.5 ml collection tube with 50 ml RNase-free water, spin 1’
- Dilute a fraction at 1:100 in 10 mM Tris pH 8 and measure concentration
Aliquot small fractions of RNA and store at -80
RNA preparation from plates with Trizol (PJ Keller)
RNA Prep Trizol (Click to Download)
Cell harvest for Western Blot
- Aspirate media from dish and scrape cells. Resuspend cells in 5ml of serum-free media or PBS and gently spin down cells.
- Aspirate supernatant and then add lysis buffer to pellet (200µl for 10cm plate or 500µl for a 15cm plate). Resuspend and leave on ice or 4°C for 30min to lyse.
- Transfer to an epi tube and spin at 13K for 15min at 4°C to pellet debris.
- At this point you can freeze the protein lysates at –20°C until use
Measure protein concentration using Lowery or Bradford assays
For Western- you will run 50µg of protein
To do this: Dilute protein if too concentrated to a volume ~20µl
Add loading dye buffer (6X) to get a 1X final concentration.
(For example add 5µl of dye buffer to 30µl of lysate…)
At this point if you want, you can store the samples at –20°C until you run the gel.
Before loading the gel, do the following to the samples:
- Boil samples (which have dye in them) for 5 min and then place IMMEDIATELY on ice.
- Quick spin the samples to collect condensation. Load gel and run.
Preparing Samples for Illumina GAIIx Sequencing (APS)
PROTOCOL: Preparing samples for Illumina Sequencing (click to download in .docx format)
Adam Skibinski/Kuperwasser Lab, Created 1-18-11
Note before starting: There are many ways to adapt libraries for sequencing; the following is one that has been successful for me and is reasonably simple. The general steps will be the same but the choice of kits, reagents etc. is not particularly important.
1. Sonication of DNA – contact Tim van Opijnen in the Camilli lab for use
1. Take an aliquot of the sample to be sequenced and dilute in 200µl of TE or water – target 5µg sample
-If you have never run the sample before, sonicate at least 1µg for several different lengths of time (e.g. 30s, 1min, 2min) and run the product on a gel until you have fragments in the range of ~200-400bp (this is what is recommended for Illumina)
2. Sonicate the sample for ~2 min (for gDNA) or ~5 min (for plasmid DNA)
3. Purify the sonicated DNA using the Qiagen PCR purification kit and elute in 32 µl of water
2. Blunting of sonicated fragments-Sonication leaves 3’ and 5’ overhangs in the sheared DNA which must be repaired for blunt-ended ligation with Illumina adaptors
1. Set up the following 50 µl reaction:
| 10x T4 DNA ligase buffer (with ATP) | 5 µl |
| 10 mM dNTP mix | 2 µl |
| T4 DNA polymerase | 2.5 µl |
| T4 Polynucleotide kinase | 2.5 µl |
| Klenow enzyme | 1 µl |
| DNA sample | 30 µl |
| Water | 7 µl |
2. Incubate 30 min at 20°C
3. Purify the product using the Qiagen PCR Purification Kit and elute in 32 µl elution buffer
3. 3’A-Tailing of Blunted Fragments- This can be done with Klenow exo (Enzymatics) but I found that Taq polymerase worked fine for this
1. Set up the following 50µl reaction:
- 30 µl blunted, sonicated DNA from step 3
- 5 µl 10x Taq buffer
- 1.5 µl 50mM MgCl2
- 2 µl 10mM dNTPs
- 11.5 µl DNase-free water
- 1 µl Taq polymerase (Invitrogen)
2. Incubate at 72°C for 30 min
3. Clean up the reaction using the Qiagen PCR Purification kit eluting in 32ul DNase-free water
4. Your product is now ready to be ligated to Illumina adapter oligonucleotides
4. Ligation — This step will TA-ligate the Illumina adapter sequences (which have 3’ T overhangs) onto the 3’-A overhangs of your DNA sample
1. Measure the concentration (in µg/ml) of your 3’A-tailed DNA using a NanoDrop spectrophotometer
2. Calculate the quantity of DNA in pmol:
Concentration (µg/ml) * Volume in ml (e.g. 0.030 ml) * 106/660 pmol/µg * 1/N,
Where N is the number of nucleotides in base pairs (suppose an average of 300). You will need twice this quantity of adapter (in pmol) in the ligation. The adapters are at 50 µM so calculate what volume you will need for that number of pmol. You will likely need to dilute the adapter to pipet a manageable volume. It is not necessary to ligate more than 10 pmol of the library as you will amplify the DNA with a PCR step.
3. Set up a ligation reaction (20 µl)
| (x) pmol DNA | varies |
| (2x) pmol Adapter | varies |
| 10x T4 ligase buffer | 2 µl |
| T4 DNA ligase | 1 µl |
| DNase-free water | to 20 µl |
4. Incubate overnight at 16°C
5. Clean-up the reaction using the Qiagen gel purification kit and elute in 32 µl water.
Note: At this point the library is adapted for Illumina sequencing and can be PCR-amplified using OLJ139 and OLJ140 as primers, available from the Tufts core, prior to submission (described below). After PCR cut out a region of the gel between 250-450 bp and submit it along with your samples. If you want to add a base-pair “tag” to differentiate samples, however, or if you want to only analyze specific sequences within your source DNA (e.g. lentiviral inserts from the TF library) then you must design specific primers to accomplish this. See “Target amplification/tagging samples” at the end of the protocol.
5. PCR Amplification
1. Set up the following reaction (50ul in a PCR tube):
| Ligated DNA from step 5 | 5 µl |
| 10 mM dNTPs | 2 µl |
| 30 µM forward primer (OLJ139) | 1 µl |
| 30 µM reverse primer (OLJ140) | 1 µl |
| 10x Taq buffer | 5 µl |
| MgCl{)2() | 10 µl |
| H2O | to 50 µl |
2. Use the following cycling parameters in a thermocycler
95°C for 2 min
95°C for 1 min
65°C for 1 min
72°C for 1 min
Repeat previous three steps x17
72°C 4 min
4°C hold
3. Run the final PCR-amplified product on a gel to make sure bands are in the range of 250-450 bp. Submit a picture of the gel along with the samples for sequencing to the Tufts core. Contact Kip Bodi for specifics on this
PREPARING SAMPLES FOR ILLUMINA
OPTIONAL STEP: Target amplification
Note: Target amplification enriches your library for a specific subset of sequences, for example lentiviral inserts from the TF library. This replaces the PCR amplification step (step 5) in the above protocol. I describe the process below in the context of the CMV promoter/V5 tag in pLenti inserts but a similar strategy could be used to amplify any set of DNAs with a common invariant sequence.
1. Design primers to amplify your sequence of interest (e.g. V5 tag)
For a 5’ constant sequence (e.g. CMV promoter):
- The forward primer will consist of ~20 bp of the 3’ portion of OLJ131 and 15 bp of the 5’ constant sequence
- The reverse primer will be OLJ140 (we have it in the lab)
For a 3’ constant sequence (e.g. V5 tag):
- The forward primer will be OLJ139
- The reverse primer should be constructed by taking 15 bp of the 3’ constant sequence followed by taking ~20bp of the 5’ portion of OLJ137, then find the reverse complement of this constructed sequence
2. PCR amplify the production – 2 step PCR
a. Set up the following reaction (50µl in a PCR tube):
| Ligated DNA from step 4 | 5 µl |
| 10 mM dNTPs | 2 µl |
| 30 µM forward primer | 1 µl |
| 30 µM reverse primer | 1 µl |
| 10x Taq buffer | 5 µl |
| MgCl2 | 10 µl |
| H2O | to 50 µl |
b. Amplify with the same parameters as step 5(2) in the above protocol
c. Transfer 5 µl to a new tube and set up another reaction:
| Ligated DNA from step 4 | 5 µl |
| 10 mM dNTPs | 2 µl |
| 30 µM OLJ139 | 1 µl |
| 30 µM OLJ149 | 1 µl |
| 10x Taq buffer | 5 µl |
| MgCl2 | 10 µl |
| H2O | to 50 µl |
3. Run on a gel and save a picture of the gel to submit with the samples. Use a NanoDrop to quantify the PCR product
TARGET ENRICHMENT
OPTIONAL STEP: “Bar-coding” samples for submission in the same lane of an Illumina sequencing reaction
Note: 4-bp “bar-codes” can be added to your adapters to distinguish different samples submitted in the same lane of the Illumina Genome Analyzer. By identifying the bar codes in the sequence data you can determine what sample each sequence came from. This is useful if you have multiple samples of low complexity (not very many different DNAs to be sequenced) and want to save $$$ but cutting down on the number of reactions. This step replaces the ligation step (step 4) in the protocol.
We have two bar-coded oligos in the lab:
- IllumAdapt_ATAT: The exact sequence of OLJ131/137 but with an additional ATAT added to the end of OLJ131
- IllumAdapt_GCGT: The exact sequence of OLJ131/137 but with an additional GCGT added to the end of OLJ131
This will allow you to submit 3 different samples at once (untagged, ATAT, and GCGT). If you need more than that you will need to have more bar codes synthesized. Talk to Adam about this, they are fancy oligos.
Replace these tagged adapters with the Illumina 131/137 adapter in step 4, ligation. Note that if you are doing target enrichment as well as barcoding you will need to account for the altered sequence in your PCR step when you design primers.
