Team:Stanford/Notebook/Lab Work/Week 5

From 2010.igem.org

(Difference between revisions)
(7/29 Thursday)
(Laura's Notebook)
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#B1006: cut with XbaI and SpeI
#B1006: cut with XbaI and SpeI
#4C5 backbone: cut with EcoRI, PstI
#4C5 backbone: cut with EcoRI, PstI
 +
for two part ligations: use GFP or RFP from #1, terminator from #2 (2 ligations- one for GFP, one for RFP)
 +
for 3a ligation: use GFP or RFP from #1, terminator from #3, 4C5 backbone from #4 (2 ligations- one for GFP, one for RFP)
==7/30 Friday==
==7/30 Friday==

Revision as of 17:50, 4 August 2010

Contents

7/26 Monday

Laura's Notebook

helped Francisco aliquot competent cells (thaw main tube- 1 mL- on ice, transfer 50 uL to each of 20 pre-chilled 0.5 mL microfuge tubes)


7/27 Tuesday

Laura's notebook

redo failed ligation: vary vector/insert ratios

prior recipe ligation 1 ligation 2 ligation 3
water none 11.0 7.0 none
vector 5.0 2.0 2.0 2.0
insert 12.0 4.0 8.0 15.0
10X buffer 2.0 2.0 2.0 2.0
ligase 1.0 1.0 1.0 1.0
  • vector=B1006 linearized (cut with EcoRI, XbaI; inserts=GFP or RFP cut with EcoRI, SpeI (6 ligations set up)
  • Francisco transformed these


7/28 Wednesday

Laura's Lab Notebook

miniprepped (ProMega kit): GFP and B1006 terminator (RFP didn't grow) NanoDrop data:

sample 260/280 260/230 ng/uL
B1006 1.61 0.95 49.4
GFP 1.83 1.22 96.4

ran 2% diagnostic gel, 75V, 1 hour

  • order: 100bp ladder, RSID1C PCR product, GFP digestion, RFP digestion, terminator digestion
  • all samples: 1 uL sample + 1 uL loading dye

ran 2% gel for gel extraction, 75V, 1 hour

  • order: 100bp ladder, GFP digestion, RFP digestion
  • 10 uL loading dye added to 50 uL digestions, 40 uL loaded on gel

gel extraction of GFP, RFP digestion products (Qiagen kit)

tube (g) tube + slice (g) gel slice (g) uL QG
GFP 1.01 1.03 0.02 60
RFP 1.01 1.03 0.02 60

ran 2% diagnostic gel of gel purified GFP and RFP digests

  • order: 100bp ladder, GFP, RFP
  • 1 uL sample + 1 uL dye + 4 uL H2O

B1006 terminator NanoDrop data (diluted 50:50 with H2O)

260/280 260/230 ng/uL
1.47 1.39 10.4
  • = 20.8 ng/uL in original sample

Digest concentrations still very low... set up larger overnight cultures- 9 mL cultures, 2 cultures each part:

  • B1006 (terminator)
  • GFP
  • RFP
  • pBAD (I0500)
  • pLUX (F2620)

7/29 Thursday

Laura's Notebook

Miniprepped (ProMega kit): RFP, GFP, B1006 terminator, F2620 (pBAD didn't grow)

  • all 18 mL (2- 9mL cultures) combined into one miniprep for each
  • Francisco did nanodrop: he has concentration data

set up digestions for both types of ligations (two-part and 3a protocol

  1. RFP and GFP: cut with EcoRI, SpeI (2 digestions each)
  2. B1006 terminator: cut with EcoRI 2 hours, heat kill 80oC 20 minutes, then add XbaI overnight
  3. B1006: cut with XbaI and SpeI
  4. 4C5 backbone: cut with EcoRI, PstI

for two part ligations: use GFP or RFP from #1, terminator from #2 (2 ligations- one for GFP, one for RFP) for 3a ligation: use GFP or RFP from #1, terminator from #3, 4C5 backbone from #4 (2 ligations- one for GFP, one for RFP)

7/30 Friday