Team:Stockholm/3 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
===Transfer of m-yCCS into pEX & Cloning of N-CPPs===
 +
Restreak results from 2/9 revealed four white (positive) clones. These were picked for colony PCR (y5, y6, y8 and y12).<br />
 +
Also picked four colonies from N-CPP plate from 30/8 (NC1, NC2, NC3 and NC4).
 +
 +
====Colony PCR====
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="2"|PCR tubes
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center" width="50"|16.2
 +
|-
 +
|DreamTaq buffer
 +
|align="center"|2
 +
|-
 +
|10 mM dNTPs
 +
|align="center"|0.4
 +
|-
 +
|pEXf
 +
|align="center"|0.4
 +
|-
 +
|pEXr
 +
|align="center"|0.4
 +
|-
 +
|DNA
 +
|align="center"|0.5
 +
|-
 +
|DreamTaq pol.
 +
|align="center"|0.08
 +
|-
 +
!Total
 +
!20 &mu;l
 +
|}
 +
 +
====Gel verification====
 +
[[image:ColPCR_NCPP_yCCS_3sep.png|200px|right|thumb|'''Gel verification of pSB1C3.N-CPPs and pEX.yCCS colony PCR.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda;=O'GeneRuler 1 kb DNA ladder]]
 +
1 % agarose, 80 V
 +
 +
'''Expected bands'''
 +
*pSB1C3.N-CPPs: 365 bp (TAT), 374 bp (LMWP), 395 bp (Tra10)
 +
*pEX.m-yCCS: 963 bp
 +
 +
'''Results'''<br />
 +
Correct bands for pEX.m-yCCS clones 5, 6 and 8. No band for y12. 5 & 8 will later be selected for plasmid prep.<br />
 +
Too large bands for all CPP clones to be correct, which is strange. New cloning will be attempted.
 +
 +
===Cloning of His&sdot;SOD into pMA===
 +
====Plasmid prep====
 +
''From 2/9 ON cultures''<br />
 +
Elution: 50 &mu;l x2
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentrations
 +
|-
 +
!Sample
 +
!Conc. [ng/&mu;l]
 +
! A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.m-yCCS 1
 +
|align="center"|143.4
 +
|align="center"|1.91
 +
|}
 +
 +
====Sequencing====
 +
pSB1C3.m-yCCS: ASB0045 A55
 +
 +
====Glycerol stock====
 +
pMA.His&sdot;SOD 2010-09-03
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
Re-ligation of 30/8 digestion.
 +
 +
====Ligation====
 +
[Dig. pSB1C3 X+A EXTR 1] = 13.72 ng/&mu;l
 +
[Dig. N-CPP X+A] = 66.6 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="2"|Ligation mix
 +
|-
 +
|Vector DNA
 +
|align="center" width="50"|6
 +
|-
 +
|Insert DNA
 +
|align="center"|9
 +
|-
 +
|5X Rapid Ligation buf.
 +
|align="center"|4
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|-
 +
!Total
 +
!20 &mu;l
 +
|}
 +
 +
====Transformation====
 +
Standard transformation procedures.
 +
#: 2 &mu;l ligation mix&dagger;
 +
#: 5 &mu;l ligation mix
 +
&dagger;: Possibly contaminated due to non-sterile transformation.
 +
 +
====Gel verification of digestion sample====
 +
[[image:Gelver dig NCPP 3sep.png|200px|right|thumb|'''Gel verification of undigested and digested (XbaI & AgeI) N-CPP cluster plasmid.'''<br />3 &mu;l &lambda;; 3 &mu;l sample<br />1 kb &lambda;=O'GeneRuler 1 kb DNA ladder; 50 bp &lambda;=GeneRuler 50 bp DNA ladder]]
 +
*NCPP: undigested N-CPP cluster plasmid
 +
*Dig NCPP: digested N-CPP cluster plasmid, XbaI and AgeI
 +
 +
1 % agarose, 100 V
 +
 +
'''Results'''<br />
 +
Difficult to interpret results, since the source plasmid size is unknown. However, it looks like the digested sample presents a shorter fragment compared to the undigested. On the other hand, it also looks like the size difference is too big to have resulted from just excising the &asymp;300 bp N-CPP cluster.<br />
 +
Also strange is that it looks like there are two bands present in the undigested sample.

Revision as of 11:12, 6 September 2010

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Contents

Andreas

Transfer of m-yCCS into pEX & Cloning of N-CPPs

Restreak results from 2/9 revealed four white (positive) clones. These were picked for colony PCR (y5, y6, y8 and y12).
Also picked four colonies from N-CPP plate from 30/8 (NC1, NC2, NC3 and NC4).

Colony PCR

PCR tubes
dH2O 16.2
DreamTaq buffer 2
10 mM dNTPs 0.4
pEXf 0.4
pEXr 0.4
DNA 0.5
DreamTaq pol. 0.08
Total 20 μl

Gel verification

Gel verification of pSB1C3.N-CPPs and pEX.yCCS colony PCR.
3 μl λ; 5 μl sample.
λ=O'GeneRuler 1 kb DNA ladder

1 % agarose, 80 V

Expected bands

  • pSB1C3.N-CPPs: 365 bp (TAT), 374 bp (LMWP), 395 bp (Tra10)
  • pEX.m-yCCS: 963 bp

Results
Correct bands for pEX.m-yCCS clones 5, 6 and 8. No band for y12. 5 & 8 will later be selected for plasmid prep.
Too large bands for all CPP clones to be correct, which is strange. New cloning will be attempted.

Cloning of His⋅SOD into pMA

Plasmid prep

From 2/9 ON cultures
Elution: 50 μl x2

DNA concentrations
Sample Conc. [ng/μl] A260/A280
pSB1C3.m-yCCS 1 143.4 1.91

Sequencing

pSB1C3.m-yCCS: ASB0045 A55

Glycerol stock

pMA.His⋅SOD 2010-09-03

Cloning of N-CPPs into pSB1C3

Re-ligation of 30/8 digestion.

Ligation

[Dig. pSB1C3 X+A EXTR 1] = 13.72 ng/μl [Dig. N-CPP X+A] = 66.6 ng/μl

Ligation mix
Vector DNA 6
Insert DNA 9
5X Rapid Ligation buf. 4
dH2O 0
T4 DNA ligase 1
Total 20 μl

Transformation

Standard transformation procedures.

  1. 2 μl ligation mix†
    5 μl ligation mix

†: Possibly contaminated due to non-sterile transformation.

Gel verification of digestion sample

Gel verification of undigested and digested (XbaI & AgeI) N-CPP cluster plasmid.
3 μl λ; 3 μl sample
1 kb λ=O'GeneRuler 1 kb DNA ladder; 50 bp λ=GeneRuler 50 bp DNA ladder
  • NCPP: undigested N-CPP cluster plasmid
  • Dig NCPP: digested N-CPP cluster plasmid, XbaI and AgeI

1 % agarose, 100 V

Results
Difficult to interpret results, since the source plasmid size is unknown. However, it looks like the digested sample presents a shorter fragment compared to the undigested. On the other hand, it also looks like the size difference is too big to have resulted from just excising the ≈300 bp N-CPP cluster.
Also strange is that it looks like there are two bands present in the undigested sample.

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