Team:Stanford/Notebook/Lab Work/Week 4

From 2010.igem.org

(Difference between revisions)
(Christopher's Notebook)
(Karina's Notebook)
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*BSA comes as 100x
*BSA comes as 100x
*made 1 mL of 1x solution by adding 100uL H20 and 100 uL BSA <br/><br/>
*made 1 mL of 1x solution by adding 100uL H20 and 100 uL BSA <br/><br/>
-
 
'''Cut the GFP and RFP lineraized plasmid at EcoRI restriction site '''
'''Cut the GFP and RFP lineraized plasmid at EcoRI restriction site '''
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Observations/Troubleshooting<br/>
Observations/Troubleshooting<br/>
no DNA bands for GFP<br/>
no DNA bands for GFP<br/>
-
* not enough DNA?
+
** not enough DNA?<br/>
-
*
+
** will double DNA concentration during digests<br/>
 +
bands are too large for RFP
==7/13 Tuesday==
==7/13 Tuesday==

Revision as of 18:24, 20 July 2010

Quad center.jpg

Spring: Brainstorming | Spring Meetings

Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries


Contents

7/12 Monday

Christopher's Notebook

  • Run Gel of Restricted Digested RBS+GFP+Terminators (I746908 partial)
  • Gel Extract
  • Run Gel of Gel Extracted Digest Products from Friday:
 3C5     E/P 2738 bp (buffer 3)
F2620   E/S (on pSB1A2) 1061 bp
  • I746908 E/P (on psb1A2) 2093 bp-can’t separate
  • B0034 X/P (on psb1A2) 12 bp-do NOT run on gel
  • J23100 E/S (on psb1A2) 35 bp-do NOT run on gel

Karina's Notebook

Goal: We couldn't extract the terminators because they were too small and ran off the gel. After discussing with Laura and Francisco, we decided to clone GFP and RFP into the plasmid in which the terminators come (pSB1AK3).

Make BSA

  • BSA comes as 100x
  • made 1 mL of 1x solution by adding 100uL H20 and 100 uL BSA

Cut the GFP and RFP lineraized plasmid at EcoRI restriction site

  • used recipe as described on July 9, 2010
  • only used 1 enzyme, therefore used 27 uL H20
  • put in waterbath at 10:30 am, and plan to take out after lunch.

Results f diagnostic gel
[insert picture here]

Observations/Troubleshooting
no DNA bands for GFP

    • not enough DNA?
    • will double DNA concentration during digests

bands are too large for RFP

7/13 Tuesday

Christopher's Notebook

  • Run Gels (0.8%) of Restriction Digests:
 1M, 2M, 2J (X/P)
 F2620 (E/S)
 3C5 (E/P)
 I0500 (E/S)

Note: for PCR of I746908: after first gel of PCR product, gel extract, and then do a restriction digest. After restriction digest, do a reaction cleanup; pieces are ready for ligation.

Laura's Notebook

  • restreaked plates from 7/1/10 (with Karina)
  • for each plate, pick 8 colonies and streak, starting at the center of the "pie piece," and working towards the perimeter of the plate
  • plate ID's:

7/14 Wednesday

Laura's Notebook

  • plates restreaked yesterday look good- bacteria grew well, some individual colonies
  • helped Francisco with minipreps (Promega kit) of : terminator, GFP, RFP
    • Francisco set up liquid cultures yesterday afternoon
  • Francisco poured, ran 0.8% gel
    • yesterday's digestions, with today's minipreps as controls
  • gel extraction: (I cut out bands, Francisco and Karina extracted)
part tube mass (g) tube + gel (g) gel (g) volume QG (uL)
terminator 1.03 1.14 0.11 330
RFP 1.11 1.18 0.07 210
GFP 1.03 1.13 0.10 300