IGEM:Harvard/2009/Lab Notebook/Week1

Monday: June 15, 2009

Tuesday: June 16, 2009

Since we are still unable to obtain PhyA, PhyB, and PIF3 cDNAs from any of the sources we have enquired with (although there is a possible lead from the Mathews group), we have decided to pursue extraction of mRNA from Arabidopsis directly. We obtained two small Colombian Arabidopsis plants from the Pierce lab, courtesy of one of their graduate students, and will use the Qiagen Plant RNeasy kit for mRNA extraction. Per their instructions, we should flash freeze the leaves with liquid nitrogen, grind them with a frozen mortor and pestal under liquid nitrogen, and then use the kit for the extraction.

As of the moment we are still waiting to hear back from the Mathews group about a cDNA library or PhyA and PhyC cDNAs; a phone call was placed to them today. Hopefully they should get back to us tomorrow.

-AG

Today we did a Midiprep on the GFP overnight cultures The midiprep procedure we used could not be found on the Qiagen website, so instead Anu googled up a midiprep protocol which has now been uploaded in the Protocols section of the Wiki but here is a summary:

1. Spin the large conical tubes that contained the culture down in a centrifuge under 4000 rpm for 10 minutes
2. Resuspend the pellet in 4mL of Buffer P1 (chilled, RNAase added - the reason it needs to be chilled is to delay the activity of the enzyme until it is added to our pellet suspension)
3. Add 4mL buffer P2, invert 4-6 times and incubate at room temperature for 5 minutes
4. Add 4mL chilled Buffer P3, mix and invert 4-6 times. Incubate on ice for 15 minutes
5. Equilibrate (synonymous to 'run through') a Qiagen Tip 100 (this can be found in the Midiprep kit box) by applying 4mL of Buffer RBT and let the column empty by gravity flow (ie. wait for the solution to completely drip from the Tip 100 thing into the tube
6. Apply the supernatant to the Qiagen tip and allow it to enter the resin by gravity flow
7. Wash the Qiagen tip with 2x 10mL Buffer QC, we run two separate 10mL QCs
8. Elute DNA with 5mL Buffer QE
9. Add isopropanol directly to the solution (not through column) to precipitate DNA. Add 3.5mL (0.7 volume) isopropanol. Mix and centrifuge immediately at 15,000g for 30min at 4°C
10. Decant supernatant. The white pellet we see is DNA.
11. Wash DNA pellet with 2mL 70% EtOH at room temperature and centrifuge at 15,000 rpm for 10 minutes
12. Precipitate with EtOH and let tube dry so we can see the DNA

This midiprep method seemed to have taken a while and the EtOH precipitation did not yield any visible pellets. So additionally we are gonig to do a backup "quick and dirty" plasmid prep from a the GFP agar plate. This procedure is just like a regular miniprep except we did not do the overnight culture and instead scraped off all the colonies from the plate to get off as much bacteria as possible to extract the maximum amount of DNA

Here are our spec results:

1. Midiprep 1 sample: ~3.8mg/μL
2. Midiprep 2 sample: ~2.2mg/μL
3. Miniprep sample: ~15.6mg/μL

We then followed up by doing digestions

GFP Vector Digest (quantities are in μL with a total volume of 40μL)

1. 30 DNA
2. 4 NEB4
3. 4 BSA
4. 1 Xba1
5. 1 Spe1

Low Promoter Digest (quantities are in μL with a total volume of 65μL)

1. 50 NA
2. 6.5 NEB4
3. 6.5 BSA
4. 1 Xba1
5. 1 Spe1

These samples were subsequently incubated at 37°C for 1 hour. We now have 3 low promoter samples and 1 GFP sample.

After the one hour incubation the samples were purified using the PCR purification kit. This removes the salts and unwanted residue, etc... and we used the Qiagen Nucleic Acid Clean up protocol

Spec results

1. Low promoter insert 1: 7.6mg/μL
2. Low promoter insert 2: 14.8mg/μL
3. Low promoter insert 3: 6.5mg/μL
4. GFP vector: 2.7mg/μL