IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-26

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Contents

New stock organization

Peng conducted an overhaul of the reagents and such. Created a new stock of rehydrated primers, and a seperate box for "gels and innoculations" which contain aliquots of low-LD-level-50%-glycerol, 1kb+ ladder, kan, dh20. Use these new reagents and hopefully contamination will go down.

Inoculation of LC1PUR1 and LC2PUR1 transformants

Yesterday's transformants grew succesfully. We inoculated 8 liquid cultures, taking 2 colonies from each of the following plates:

  • LC1PUR1 3kb #1
  • LC1PUR1 400b #2
  • LC2PUR1 3kb #1
  • LC2PUR1 400b #2

The liquid medium consisted of:

  • 8 µL LB-min (We used LB-min because we ran out of regular LB)
  • 3.2 µL Kanamycin

Sequencing

We figured out the molarities of the M13F and M13R primer solutions that Invitrogen provides with its Zero Blunt Topo Cloning kit. They are 20.31 µM and 19.18 µM, respectively. With this info in hand, we completed the sequencing reactions started yesterday, filled an order with GeneWiz, and sent them off.

Miniprep of HH6

Miniprepped last night's HH6 re-inoculations. Followed Qiagen centrifuge miniprep protocol. Addtional note: used EB buffer for DNA elution instead of dH2O.

Ended up with about 100µL of DNA split between 2 tubes.

Nanodropped the minipreps and found that the DNA concentrations are pretty low, around 5.4 ng/µL.

Primer diagnostic gel

Click for legend
Click for legend

Because of the smearing in the gel from yesterday's mutagenesis attempt, we suspected that our primers might be contaminated. To investigate this problem, we made new dilutions of our primers from the master stocks, and ran 4 of them on a gel along with 4 of the old primer mixes. We did the same with our dNTPs.

The gel was 1.5% agarose, run for 45 minutes. See image at right.

Our gel did not detect any contamination in our primers. This doesn't necessarily mean they're not contaminated; PCR can amplify contamination that our gel would not pick up.

Notice that there appear to be no bands in lanes 8 and 9 (old and new EcoR1). We will nanodrop our primer mixes to check the DNA concentrations.

Update: We checked the concentration of EcoR1 in the nanodrop and it appears to be present (~190 ng/µL), so we mostly likely made a mistake in loading the gel for lanes 8 and 9.

Rerun of mutagenesis gel

In a separate attempt to reduce smearing, Peng reran some of the products from yesterday's mutagenesis PCR on a thicker gel for a longer time (1.5% agarose, 2 hours). Image to the right. The results look the same as yesterday's, although there are two noticable differences.

  • spearing is a bit lower
  • there is not a 300bp band caused by BTB loading dye
Was run this time at 60V for 2h on a 1.5% gel.
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