Team:Stanford/Notebook/Lab Work/Week 3

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==7/12 Monday==
 +
===Christopher's Lab 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). <br/><br/>
 +
 +
'''Make BSA'''
 +
*BSA comes as 100x
 +
*made 1 mL of 1x solution by adding 900uL H20 and 100 uL BSA <br/><br/>
 +
 +
'''Cut the GFP and RFP lineraized plasmid at EcoRI restriction site '''
 +
*used recipe as described on [https://2010.igem.org/Team:Stanford/Notebook/Lab_Work/Week_3 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. <br/><br/>
 +
 +
'''Results of diagnostic gel''' <br/>
 +
[insert picture here]<br/>
 +
 +
''Observations/Troubleshooting'':<br/>
 +
*no DNA bands for GFP<br/>
 +
** not enough DNA?<br/>
 +
** will double DNA concentration during digests<br/>
 +
*bands are too large to be RFP
 +
**possible partial digest?
 +
**leave digests overnight
 +
<br/>
 +
 +
'''Digests'''<br/>
 +
Digest miniprepped GFP, RFP and Terminators.<br/>
 +
*Cut GFP and RFP at E and S
 +
*Cut terminators at E and X<br/><br/>
 +
''Recipe''<br/>
 +
25 uL DNA<br/>
 +
1.0 uL enzyme 1<br/>
 +
1.0 uL enzyme 2<br/>
 +
5.0 uL NEB Buffer 2<br/>
 +
5.0 uL BSA (10x) <br/>
 +
13 uL H20<br/><br/>
 +
Total Volume: 50 uL<br/>
 +
Run digests overnight
 +
 +
==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
 +
|}
 +
 +
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Revision as of 22:35, 21 July 2010

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Contents

7/12 Monday

Christopher's Lab 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 900uL 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 of 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 to be RFP
    • possible partial digest?
    • leave digests overnight


Digests
Digest miniprepped GFP, RFP and Terminators.

  • Cut GFP and RFP at E and S
  • Cut terminators at E and X

Recipe
25 uL DNA
1.0 uL enzyme 1
1.0 uL enzyme 2
5.0 uL NEB Buffer 2
5.0 uL BSA (10x)
13 uL H20

Total Volume: 50 uL
Run digests overnight

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



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