Note: this is a search engine friendly version of my lab notebook, please see the pdf version of this document for a more human friendly, printer friendly version.

Appendix D
Equipment

D.1  DNA work

Please see the pdf version for figures Figure D.1: We have two BioRad PCR machine both with two 48-well blocks. If life were to run my way, we'd have one with a 96-well block and one with a 384-well block. Brief Update Sat Jul 29 20:14:37 EDT 2006:   Life is now running my way with regards to PCR machines. We now have a 384-well and a 96-well block. Gear that works in them is (all from BioRad): MLP-9601 unskirted PCR plate (for 96-well PCR min vol 10 ml ), MSB-3842 (for 384-well min vol 5 ml ), MSB-1001 microseal 'B' adhesive seals (for the top of either plate).

D.1.1  qPCR

The following instructions are for the 7900HT. The template was setup by David Lorenz in Jim Collin's lab.

Josh Thaden's 12 steps to qPCR success

1. spin plate (1000 RPM for > = 2 min) IN A ROOM TEMPERATURE CENTRIFUGE⁵⁷
2. log into computer log: collins, pwd: genetream
3. open PCR-w-melt icon
4. define wells and click Use
5. set sample volume (instrument tab, to the right of the define wells tab)
6. save data in appropriate folder
7. click Connect (instrument tab in the main menu) ⁵⁸
8. Click open/close button in Tools menu; Load 384-well plate
9. Click open/close button (to close door of machine)
10. press Start in instrument tab
11. software inferace will show remaining time after a couple minutes
12. Save two files (go to Export): results table (just Ct values), clipped (fluorescent values at each time)

D.2  Gels

D.2.1  MiniGels

Protein

Run using premade gels from invitrogen using their sure-lock system.

Nucleic Acid

Run using fisher mini gel rigs

D.2.2  Big Gels

We have an Owl Gater A1 gel rig (the owl rigs are very nice).

gel thickness (cm)	0.25	0.5	0.75	1.0
volume to achieve thickness above (ml)	81	163	244	325
1.0 mm, 25 well comb volume (ml )	2	7	12	17
1.5 mm, 25 well comb volume (ml )	3	11	18	26
1.5 mm, 9 well comb volume (ml )	8	28	49	69

Agarose gels are run

D.2.3  Polyacrylamide pre-cast Gels

I use the ones from invitrogen:

well/comb type	maximum volume
5 well, 1.0 mm	60 ml
8 well, 1.0 mm	28 ml
10 well, 1.0 mm	25 ml
10 well, 1.5 mm	37 ml
12 well, 1.0 mm	20 ml
15 well, 1.0 mm	15 ml
15 well, 1.5 mm	25 ml

For TBE Gels I use Hi-Density Sample Buffer (5x) for 6% gels the bromophenol blue (dark blue) dye band is at 65 bp and the xylene cyanol band (blue green) is at 250 bp. I run the 6% TBE gel at 200V for (30-90) minutes. For TBE Urea gels I use Novex TBE Urea Sample buffer and I flush the wells a few times with 1x TBE running buffer to remove Urea. I also heat the samples for 3 minutes at 70^° C after mixing the sample with the loading dye. The recommend not more than 200 ng DNA / band. Use standard TBE running buffer. I use the elution buffer mentioned in Science Shendure et.al. polony paper to remove the DNA from cut bands (this is pretty similar to the sambrook method, Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method protocol 12, 5.51): dice fragments (I use the tube inside a tube method, NOT the razor blade technique they mention). Then I add PAGE elution buffer to the diced pieces: 10 mM Tris, 50 mM NaCl, 1 mM EDTA. Place at 37 C overnight. Next day spin down 1 min maximum speed. To improve yield wash gel framents with an additional 200 ml of elution buffer. Purify with phenol:chloroform. Below is taken from sambrook and the invitrogen novex quick reference card (a * indicates invitrogen sells that size and that I took the dye migration numbers form invitrogen) the number in parenthesis indicates the TBE-Urea dye migration band (see also Figure ):

concentration of acrylamide	effective range of separation (bp)	bromophenol blue (dark)	xylene cyanol (light)
3.5%	1000-2000	100	460
5%	80-500	65	260
* 6%		65 (25)	250 (110)
* 8%	60-400	25	220
* 10%		35 (20)	120 (55)
12%	40-200	20	70
* 15% (urea only)	25-150	15 (10)	60 (40)
* 20%	6-100	15	50

Please see the pdf version for figures Figure D.2: Gel migration chart for the novex gels.

D.3  Imaging

Gels are imaged on a Versadoc imaging system.

D.4  Weighing, Measuring, pH

Please see the pdf version for figures Please see the pdf version for figures Figure D.3: We have two scales. The Denver (a) is for low precision stuff and the Toledo is for higher precision weighing. I use the Toledo for anything under a gram. If I were to buy a low precision scale. I wouldn't buy this Denver, it maxes out at 200g. If you have a deep-well plate that you are having trouble balancing on the centrifuge it can easily weigh more than this. I get a scale that goes up to 5kg even if I had to give up a little precision Please see the pdf version for figures Figure D.4: The Denver 200 pH meter does the trick

D.5  Growing stuff and warming stuff

Please see the pdf version for figures Figure D.5: This Thermo electron shaking incubator is very quiet but only works well at temperatures 35C and above. It is also very slow to start and stop and has an annoyingly long delay between when you push the start/stop button and when it decides "ok I'll begin stopping now". We have two stackable ones.

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Footnotes:

¹linear extrapolation ²just discovered [Mon Oct 17 16:05:30 EDT 2005] dctR is actually the same as yhiF so it's not missing ³this is nice because normally when I do statistical tests I'm looking to improve something; now I just want to make sure things aren't getting any worse ⁴I spilled a little of lrpB3 here :( Only a few microliters though. ⁵had strong white precipitate. would assume to be DNA but wasn't in previous etOH prep. I might need a phenol/chloroform step to prevent this :( ⁶unlike that protocol I used EC broth because I was out of LB ⁷had strong white precipitate. would assume to be DNA but wasn't in previous etOH prep. I might need a phenol/chloroform step to prevent this :( ⁸I originally wanted to use 5 mg , but sample 2 wasn't concentrated enough for me to do that and still be under the max volume of the standard Superscript II protocol (20 ml ). ⁹35 ml was chosen because it allows 1 ml to be used to spec the DNA and the remaining amount is the maximum allowable volume for the end-repair kit ¹⁰8.07 ml of sample 3 and 8.16 ml of sample 4 ¹¹if I had to do it over again, I'd add another 0.25 ml of RNAse H here ¹²35 ml was chosen because it allows 1 ml to be used to spec the DNA and the remaining amount is the maximum allowable volume for the end-repair kit ¹³I didn't take the sample to run on the gel until after the adaptor ligation step below. ¹⁴Note: this didn't go exactly as planned. After 12 hours the temperature went on hold at 4^° C (i.e. I didn't immediately heat inactivate the ligase, but 4^° C should've slowed it down a bit). The sample remained at this temperature for approximately 5 hours before I went to the next step. ¹⁵this shouldn't be necessary, but I was afriad that perhaps the ATP would've been exhausted from the long ligation ¹⁶Last time I resuspended into 50 ml , which is better for the DNA-free kit. However, the MICROBExpress kit allows at most 10 mg in 15 ml . With 50 ml it wouldn't have been concentrated enough to get 10 mg in such a small volume. I want to make sure and maximize the starting material, because so much RNA is lost after the rRNA removal (final yield from 10 mg is expected to be 1-2.5 mg ). ¹⁷I don't know how well this works, but it was fun to play with magnetic beads for the first time. ¹⁸the invitrogen protocol recommends much less starting material for mRNA than for total RNA; 3.3 ml of sample 5 and 3.1 ml of sample 6 ¹⁹if I had to do it over again, I'd add another 0.25 ml of RNAse H here ²⁰35 ml was chosen because it allows 1 ml to be used to spec the DNA and the remaining amount is the maximum allowable volume for the end-repair kit ²¹Note: this didn't go exactly as planned. After 12 hours the temperature went on hold at 4^° C (i.e. I didn't immediately heat inactivate the ligase, but 4^° C should've slowed it down a bit). The sample remained at this temperature for approximately 5 hours before I went to the next step. ²²this shouldn't be necessary, but I was afriad that perhaps the ATP would've been exhausted from the long ligation ²³In the invitrogen manual for making cDNA they suggest 200 U (200 U = 1 ml per mg of RNA) ²⁴As I've done previously with the BamHI adaptors, I'll phosphorylate the adaptors AFTER they've been ligated to the RNA. It's not really as important with the NON-palandromic adaptor, but since I already had an unphosphorylated adaptor, I'll stick with the post-phosphorlation method. ²⁵the long piece is the same I used before ²⁶I know the MmeI deactivation temp is 80C for 10 min, but I was worried about melting the DNA. Probably wouldn't affecting anything by going to 80C, but who knows. ²⁷my piece at 80bp is a little below the removal limit for the PCR kit, but a little above the limit for the gel cleanup kit. Hopefully I don't lose too much. I can try this real easy with the RCA sample I have. ²⁸this is the same 80mer described in section on page ²⁹see sections 6.6.2.1 and 6.6.2.1 starting on page pageref ³⁰she claims this is way to high and you can't ever get your captured oligo off ³¹I used 567 ng of the RCA product ³²had to add 20 ml TE to the dried membrane to elute ³³mostly there were small deletions ³⁴I did purposely cut a tiny-bit inside the PET band to lessen the amount of non-PET DNA, maybe the Qubit dye is more sensitive to longer DNA? ³⁵I realize it is more parsimonious to add pdhR gene and then the two promoters, but the pdhR gene has a cutter in the middle of if that is needed to clone the two promoters. ³⁶I considered the promoter to be the first 200 bp upstream of the ATG start codon ³⁷This is the first time I've used Sybr Safe. It is very clear on the transilluminator, much better than EtBr. ³⁸from the total 30 ml elution volume of the Qiagen kit ³⁹I would have preferred to check them by PCR, but I worgot to buy the primers to check these things with ⁴⁰I use 1000 here because the cells have plenty of time to grow before the next morning ⁴¹Tim's toggle is currently (Green / No Green) but there's no reason it couldn't be (Green / Red) ⁴²LB fluoresces way to much in the yellow and orange area, making it hard to see the proteins in LB ⁴³80 ml was just enough space for the entire sample ⁴⁴if doing blue-white screening, add 40 ml of Blue-White Select Screening Reagent [Sigma] 10 min before plating the cells ⁴⁵if you don't want really sheared DNA try to be gentle with your resuspension (i.e. don't vortex the crap out of the cells) ⁴⁶this is the fastest our centrifuge will go, the actually protocol says 6000 rpm for 10 min ⁴⁷not all, but most of the cells will be pelleted; solution is still cloudy, so longer spin times might bring up the final DNA yield ⁴⁸the original protocol reads: (optional) Resuspend the cells in 20 ml of H2O. Spin again. ⁴⁹original protocol recommends 10 min at 10,000 rpm ⁵⁰make sure the entire tube surface is coated with Soln II ⁵¹In the past, I resuspended into 50 ml , which is better for the DNA-free kit. However, the MICROBExpress kit allows at most 10 mg in 15 ml . With 50 ml it wouldn't have been concentrated enough to get 10 mg in such a small volume. I want to make sure and maximize the starting material, because so much RNA is lost after the rRNA removal (final yield from 10 mg is expected to be 1-2.5 mg ). ⁵²this is more than Invitrogen recommends for mRNA, but this is important to have enough cDNA for downstream steps. Note that 11 ml is as much RNA as you can add, so it helps to have it really concentrated. ⁵³add 200 U (1 ml ) of Superscript III per 1 mg of mRNA ⁵⁴35 ml was chosen because it allows 1 ml to be used to spec the DNA and the remaining amount is the maximum allowable volume for the end-repair kit ⁵⁵in case the ATP has been exhausted from the long ligation ⁵⁶we are adding phosphates to the adaptors ⁵⁷if the centrifuge is not at ambient temperature, you'll get condensation on all of your wells; for this reason, most users prop open the lid on the centrifuge in the qPCR room ⁵⁸if you are using the hs dynamo kit from NEB, you need to switch to 15 minutes initial heat denaturation