IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-7

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Note: a backslash '\' followed by a pair of letters indicates a digest, e.g. \X-P means an XbaI-PstI digest.

Contents

T-shirt design


To-do

  • Miniprep the 15 ligation transformants we grew up yesterday (10 KaiA+bkb, 5 KaiB+bkb)

LacIq and J04450 (RFP insert)

There appears to be constitutive RFP expression, despite the use of lacIq-carrying competent cells (Top10F')
There appears to be constitutive RFP expression, despite the use of lacIq-carrying competent cells (Top10F')


In today's meeting, George Church recommended putting lacIq on the plasmid.

Gel purification of J04450, high-copy vector, and pSB4A3 low-copy vector

We gel-purified the J04450, high-copy vector, and psB4A3 low-copy vector that we extracted from yesterday's gel.

Miniprep of 15 samples from the KaiA+bkb and KaiB+bkb transformation

15 samples (KaiA+bkb 2-11, KaiB+bkb 2-6) miniprepped from 8uL of culture; made glycerol stock also. Both are sitting in their seperate containers in the freezer or -80 respectively. Nanodrop values varied from 10-20ng/uL - next time only grow in 2uL LB?

Digest X-P to test ligation

KaiA+bkb 1-11 and KaiB+bkb 1-6 were digested with X&P to see if the ligation worked or failed. The protocol based loosely on the Silver protocol, but assuming each sample has ~15ng/uL DNA, is:

  • 19.25 uL DNA (~15 ng/uL gives 0.288ug DNA)
  • 2 uL H2O
  • 2.5 uL buffer (EcoRI buffer or Buffer 3)
  • 0.5 uL enzyme 1 (XbaI)
  • 0.5 uL enzyme 2 (PstI)
  • 0.25 uL BSA

25 uL total volume.

MM:

  • 19x2=38 dh20
  • 19x2.5= 47.5 buffer 3
  • 19x0.5 = 9.5 XbaI
  • 19x0.5 = 9.5 PstI
  • 19x0.25 = 4.75 BSA

pipet 5.75 into each.

Ligation of J04450 + pSB4A3

We iigated the gel-purified J04450 (\X-P) and pSB4A3 (\E-S) using the Roche rapid ligation kit.

Reaction:

  • 2 uL pSB4A3
  • 4 uL J04450
  • 2 uL DNA dilution buffer (vial 2)
  • 2 uL H2O
  • 10 uL DNA ligation buffer (vial 1)
  • 1 uL ligase (vial 3)

Transformation of J04450 + pSB4A3 into Top10 and Top10F'

We transformed the J04450 + pSB4A3 ligation into Top10 and Top10F' cells, to test for constitutive expression of RFP.

Transformations:

  • 30 uL Top10 competent cells, 3 uL ligation
  • 30 uL Top10F' competent cells, 3 uL ligation
  • 10 uL Top10 competent cells, 1 uL positive control
  • 10 uL Top10F' competent cells, 1 uL positive control
  • 10 uL Top10 competent cells, 3 uL H2O
  • 10 uL Top10F' competent cells, 3 uL H2O

Plated on 8 LB-carb plates: 1 for each of the controls, and 2 for each of the ligation transformations.

Digests

The digests that were completed earlier in PCR machines 1 and 2 (from earlier) were run for 2 hours with a 20 minute heat shock to inactivate the restriction enzymes.

The digests were then run on 1% agarose gel that contained 20 lanes (25uL capacity)

Gels

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Analysis

It seems like we got KaiA but not KaiB, from the digests above. When the plasmids are cut with only one of the two restriction enzymes (due to a backwards insert in the vector), there should be a band about 3kb; that is seen in every lane. Interestingly, KaiA (which is 855 bp) shows up on the gel as an 850 bp band, but the vector containing KaiA should have been 2 kb instead of 3 kb.

Because we feel that we should analyze this further, we will wait until the morning to figure out what to do.
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