Team:Stanford/Notebook/Lab Work/Week 5

From 2010.igem.org

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(→Laura's Lab Notebook)
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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
 +
|-
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|1.47 || 1.39 || 10.4
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|}
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*= 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==
==7/29 Thursday==

Revision as of 23:39, 3 August 2010

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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

7/30 Friday

Retrieved from "http://2010.igem.org/Team:Stanford/Notebook/Lab_Work/Week_5"