Team:Stockholm/3 September 2010

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{{Stockholm/Top2}}
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==Andreas==
==Andreas==
===Transfer of m-yCCS into pEX & Cloning of N-CPPs===
===Transfer of m-yCCS into pEX & Cloning of N-CPPs===
Line 47: Line 48:
Too large bands for all CPP clones to be correct, which is strange. New cloning will be attempted.
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====
 
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''From 2/9 ON cultures''<br />
 
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Elution: 50 &mu;l x2
 
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{|border="1" cellpadding="1" cellspacing="0"
 
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!colspan="3"|DNA concentrations
 
-
|-
 
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!Sample
 
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!Conc. [ng/&mu;l]
 
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! A<sub>260</sub>/A<sub>280</sub>
 
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|-
 
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|pSB1C3.m-yCCS 1
 
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|align="center"|143.4
 
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|align="center"|1.91
 
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|}
 
-
 
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====Sequencing====
 
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pSB1C3.m-yCCS: ASB0045 A55
 
-
 
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====Glycerol stock====
 
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pMA.His&sdot;SOD 2010-09-03
 
===Cloning of N-CPPs into pSB1C3===
===Cloning of N-CPPs into pSB1C3===
Line 115: Line 95:
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 />
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.
Also strange is that it looks like there are two bands present in the undigested sample.
 +
 +
===Cloning of SOD&sdot;His into pMA===
 +
====Sequencing results====
 +
*SH1: pMA.SOD&sdot;His 1 ([[media:SH1_premix.txt|fasta]])
 +
*SH2: pMA.SOD&sdot;His 2 ([[media:SH2_premix.txt|fasta]])
 +
 +
Ran nucleotide Blasts (Blastn) to verify the sequencing results ([[media:Blastn_SH1_premix-pMA.SOD*His_3sep.txt‎|SH1 Blastn]] / [[media:Blastn_SH2_premix-pMA.SOD*His_3sep.txt‎|SH2 Blastn]]). Both Blasts revealed three identical silent mutations within the His tag. However, since they don't affect the amino acid sequence, and don't result in any rare codons, no action will be needed.
 +
 +
== Mimmi ==
 +
 +
=== Saftey issues on the home page ===
 +
 +
*Making a first layout to answer the saftey questions...
 +
 +
 +
=== pMA.his.SOD ===
 +
 +
*Plasmid prep. following E.Z.N.A. plasmid mini kit protocol 1
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**Wash two times with DNA wash buffer
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**Eluate in 50µl sH<sub>2</sub>O
 +
 +
 +
'''DNA concentration'''
 +
 +
266ng/µl
 +
 +
 +
 +
=== MITF-M ===
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==== Colony PCR ====
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 +
Fermentas Pfu
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
| x5
 +
| rowspan="9" width="100" |
 +
! Primers
 +
| rowspan="9" width="100" |
 +
! colspan="2" | conditions
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| rowspan="3" |
 +
|-
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| sH<sub>2</sub>O
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| 17
 +
| 85
 +
| pSB_VF2
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! time
 +
! &deg;C
 +
|-
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| 10x buffer
 +
| 2.5
 +
| 12.5
 +
| pSB_VR
 +
| 2m
 +
| 95
 +
|-
 +
| dNTP
 +
| 2.5
 +
| 12.5
 +
| rowspan="7" |
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| F primer
 +
| 1
 +
| 5
 +
| 30s
 +
| 55
 +
| > 30 cycles
 +
|-
 +
| R primer
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| 1
 +
| 5
 +
| 2m40s
 +
| 72
 +
| )
 +
|-
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| DNA
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| 0.5
 +
| 5x0.5
 +
| 10m
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| 72
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| rowspan="2" |
 +
|-
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| Pfu pol
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| 0.5
 +
| 2.5
 +
| oo
 +
| 10
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
| 125µl
 +
|}
 +
 +
 +
 +
=== pMA.his.SOD ===
 +
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==== Digestion ====
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{|
 +
! Mix
 +
| (µl)
 +
| (µl)
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| rowspan="7" width="100" |
 +
! colspan="2" | Conditions
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| rowspan="7" width="100" |
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| [pMA.SOD.his] = 246ng/µl -> ~2µg = 8µl
 +
|-
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| DNA
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| 8
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| 7.5
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! Time
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! &deg;C
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| [pMA.his.SOD] = 280ng/µl -> ~2µg = 7.5µl
 +
|-
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| s<sub>2</sub>O
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| 17
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| 17.5
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| 30m
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| 37
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| [pSB1C3] = 148ng/µl -> ~1.2µg = 8µl
 +
|-
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| 10x buffer
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| 3
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| 3
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| 20m
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| 65
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| rowspan="4" |
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|-
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| XbaI
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| 1
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| 1
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| rowspan="3" colspan="2" |
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|-
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| PstI
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| 1
 +
| 1
 +
|-
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| align="right" | tot
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| 3x30µl
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|}
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==== Gel ====
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[[Image:2010-09-03_SOD.his_+_his.SOD_+_pSB1C3_X+P.jpg|100px|thumb|left|]]
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{|
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! well
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! sample
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|-
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| 1
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| 1kb ladder
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|-
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| 2
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| pMA.SOD.his
 +
|-
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| 3
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| pMA.his.SOD
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|-
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| 4
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| pSB1C3.RFP
 +
|}
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 +
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*Cut gel bands
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*Save in fridge
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 +
{{Stockholm/Footer}}

Latest revision as of 10:59, 26 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


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 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.

Cloning of SOD⋅His into pMA

Sequencing results

  • SH1: pMA.SOD⋅His 1 (fasta)
  • SH2: pMA.SOD⋅His 2 (fasta)

Ran nucleotide Blasts (Blastn) to verify the sequencing results (SH1 Blastn / SH2 Blastn). Both Blasts revealed three identical silent mutations within the His tag. However, since they don't affect the amino acid sequence, and don't result in any rare codons, no action will be needed.

Mimmi

Saftey issues on the home page

  • Making a first layout to answer the saftey questions...


pMA.his.SOD

  • Plasmid prep. following E.Z.N.A. plasmid mini kit protocol 1
    • Wash two times with DNA wash buffer
    • Eluate in 50µl sH2O


DNA concentration

266ng/µl


MITF-M

Colony PCR

Fermentas Pfu

Mix (µl) x5 Primers conditions
sH2O 17 85 pSB_VF2 time °C
10x buffer 2.5 12.5 pSB_VR 2m 95
dNTP 2.5 12.5 30s 95 )
F primer 1 5 30s 55 > 30 cycles
R primer 1 5 2m40s 72 )
DNA 0.5 5x0.5 10m 72
Pfu pol 0.5 2.5 oo 10
tot 25µl 125µl


pMA.his.SOD

Digestion

Mix (µl) (µl) Conditions [pMA.SOD.his] = 246ng/µl -> ~2µg = 8µl
DNA 8 7.5 Time °C [pMA.his.SOD] = 280ng/µl -> ~2µg = 7.5µl
s2O 17 17.5 30m 37 [pSB1C3] = 148ng/µl -> ~1.2µg = 8µl
10x buffer 3 3 20m 65
XbaI 1 1
PstI 1 1
tot 3x30µl


Gel

2010-09-03 SOD.his + his.SOD + pSB1C3 X+P.jpg
well sample
1 1kb ladder
2 pMA.SOD.his
3 pMA.his.SOD
4 pSB1C3.RFP


  • Cut gel bands
  • Save in fridge





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/3_September_2010"