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

(Difference between revisions)
 Revision as of 11:04, 21 July 2012 (view source) (→Ligation of pSC002 dig. + pSC003 dig.)← Previous diff Current revision (16:53, 22 July 2012) (view source) (→Transformation of the Ligation) (4 intermediate revisions not shown.) Line 67: Line 67: (*) : calculated thanks to the following formula : (*) : calculated thanks to the following formula : - $Weight_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Weight_{vector}$ + $Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector}$ - ratio being 5 (5 times as many insert as vector). + ratio being 5 (5 times more insert than vector). -> 22°C -> 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 + + + __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.

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