IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/07/21

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(→Miniprep of : pSC001a, pSC002a&b, pSC003a)
Current revision (16:53, 22 July 2012) (view source)
(→Transformation of the Ligation)
 
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== Migration on 1% agarose gel ==
== Migration on 1% agarose gel ==
 +
Migration of 10µL of the digestions
 +
[[Image:Gel_20_07_Digestion.png|thumb|center|500px|Agarose gel of the previous digestion. E : EcoRI; P: PstI; X : XbaI; S : SpeI; Ø : no enzyme.]]
-
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
+
The bands are as expected, we can PCR purif the digestion (2 tubes of 30 µL)
 +
 
 +
== PCR purif ==
 +
 
 +
The PCR purif is made with the promega kit, I recover dna in 30 µL of water.
 +
 
 +
* pSC001 : 83 ng/µL
 +
* pSC002 : 76 ng/µL
 +
 
 +
-> good.
 +
 
 +
== Ligation of pSC002 dig. + pSC003 dig. ==
 +
 
 +
* vector (pSC002) : 1 µL (ie : 76ng)
 +
* insert (pSC003) : 37 µL (*)
 +
* buf. 10X : 1µL
 +
* T4 dna ligase : 1µL
 +
* water : 4 µL
 +
 
 +
(*) : calculated thanks to the following formula :
 +
 
 +
<math>Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector}</math>
 +
 
 +
ratio being 5 (5 times more insert than vector).
 +
 
 +
-> 22°C
 +
 
 +
* About 4h later : 10min at 65°C, to deactivate the T4 dna ligase.
 +
 
 +
== Transformation of the Ligation ==
 +
 
 +
* Standard protocol, with the following quantities :
 +
 
 +
{| border = "1" width="45%"
 +
! width="35%" | What's in the tube
 +
! width="5%" | DNA volume (µL)
 +
! width="5%" | Cell volume (µL)
 +
! width="8%"| Plates (LB + Amp)
 +
|-
 +
 +
|Ligation
 +
|10
 +
|40
 +
|180µL pur & 200µL 1/10
 +
|-
 +
|positive transformation control (pSB1A2::R0011)
 +
|0,5
 +
|20
 +
|200µL
 +
|-
 +
|negative ligation control (pSC002 dig. ie. insert)
 +
|1
 +
|20
 +
|200µL
 +
|-  
 +
|negative ligation control (pSC003 dig. ie. vector)
 +
|1
 +
|20
 +
|200µL
 +
|-
 +
|negative transformation control (NEB only)
 +
|0
 +
|20
 +
|200µL
 +
|-  
|}
|}
 +
 +
--> Plate at 37°C O/N
 +
 +
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
 +
__NOTOC__
__NOTOC__
[[category:OWWLabNotebookV1]]
[[category:OWWLabNotebookV1]]

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Making -20°C & -80°C stocks

  • I saved 750µL of O/N culture, in order to add 250µL of 60% glycerol sterile, in sterile tubes. I don't find them. I put the 750µL in normal eppendorf, at 4°C.

Miniprep of : pSC001a, pSC002a&b, pSC003a

  • I used 500µL of Lysis solution instead of 250µL
  • Recover plasmid in 70µL of water.
  • Nanodrop :
    • pSC001a : 186,4 ng/µL
    • pSC002a : 157,1 ng/µL
    • pSC002b : 144 ng/µL
    • pSC003a : 142,4 ng/µL

->the big amount of lysis solution did not perturb the miniprep.

Digestion of pSC001(a) & pSC002(a)

  • pSC001 : SpeI + PstI
    • DNA : 20 µL / 3
    • SpeI : 2 µL / 0
    • PstI : 2 µL / 0
    • buf. 10X : 4 µL / 2
    • water : 12 µL / 15
  • pSC002 : EcoRI + XbaI
    • DNA : 20 µL / 3
    • EcoRI : 2 µL / 0
    • XbaI : 2 µL / 0
    • buf. 10X : 4 µL / 2
    • water : 12 µL / 15

--> 30 min, 37°C water bath.

Migration on 1% agarose gel

Migration of 10µL of the digestions

Agarose gel of the previous digestion. E : EcoRI; P: PstI; X : XbaI; S : SpeI; Ø : no enzyme.
Agarose gel of the previous digestion. E : EcoRI; P: PstI; X : XbaI; S : SpeI; Ø : no enzyme.

The bands are as expected, we can PCR purif the digestion (2 tubes of 30 µL)

PCR purif

The PCR purif is made with the promega kit, I recover dna in 30 µL of water.

  • pSC001 : 83 ng/µL
  • pSC002 : 76 ng/µL

-> good.

Ligation of pSC002 dig. + pSC003 dig.

  • vector (pSC002) : 1 µL (ie : 76ng)
  • insert (pSC003) : 37 µL (*)
  • buf. 10X : 1µL
  • T4 dna ligase : 1µL
  • water : 4 µL

(*) : calculated thanks to the following formula :

Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector}

ratio being 5 (5 times more insert than vector).

-> 22°C

  • About 4h later : 10min at 65°C, to deactivate the T4 dna ligase.

Transformation of the Ligation

  • Standard protocol, with the following quantities :
What's in the tube DNA volume (µL) Cell volume (µL) Plates (LB + Amp)
Ligation 10 40 180µL pur & 200µL 1/10
positive transformation control (pSB1A2::R0011) 0,5 20 200µL
negative ligation control (pSC002 dig. ie. insert) 1 20 200µL
negative ligation control (pSC003 dig. ie. vector) 1 20 200µL
negative transformation control (NEB only) 0 20 200µL

--> Plate at 37°C O/N

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