Team:HokkaidoU Japan/Notebook/aggregation Week 5

From 2012.igem.org

(Difference between revisions)
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Our competent cell Protocol
Our competent cell Protocol
#Single colony isolation on LB plate
#Single colony isolation on LB plate
-
#incubate the plate for 15-19 hours at 37℃
+
#incubated the plate for 15-19 hours at 37℃
-
#lift colony of E.coli into 2 ml LB
+
#lift colony of E. coli into 2 ml LB
-
#culture cells at 37℃ for 12-16 hours at 180-200 rpm
+
#cultured cells at 37℃ for 12-16 hours at 180-200 rpm
-
#transfer 30 ul, 100 ul, 300 ul of the culture into 100 ml of SOB medium, respectively
+
#transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB , respectively
-
#culture cells at 20℃ (for 24 hours over) at 180-200 rpm (to ΔOD550nm=0.5～0.6)
+
#cultured cells at 20℃ (for 24 hours over) at 140 rpm
-
#leave the 300 ml flask for 10 min on ice
+
#selected culture by measuring OD 600
-
#transfer the culture into two 50 ml Falcon tube
+
#left the 300 ml flask for 10 min on ice
-
#centrifuge 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
+
#transfered the culture into two 50 ml Falcon tube
-
#suspend the pellet in ice-cold 15 ml of TB (Transformation Buffer)(7.5 ml/tube)
+
#centrifuged 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
-
#collect them in one tube
+
#suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer)(7.5 ml/tube)
-
#centrifuge 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
+
#collected them in one tube
-
#suspend the pellet in ice-cold 3.2 ml of TB (Transformation Buffer)
+
#centrifuged 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
-
#Instil 0.24 ml of DMSO in precipitant
+
#suspended the pellet in ice-cold 3.2 ml of TB
-
#leave the 50 ml Falcon tube for 10 min on ice
+
#Instiled 0.24 ml of DMSO in precipitant
-
#divide 50ul of solutions in each 1.5 or 0.5 ml tubes
+
#left the 50 ml Falcon tube for 10 min on ice
-
#Freeze the suspension in liquid nitrogen
+
#divide 50ul of solutions in each 0.5 ml tubes
-
#store at –80℃
+
#Freezed the suspension in liquid nitrogen
 +
#stored at –80℃
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[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|digestion result]]
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|digestion result]]
There were low concentration band in above of thin band. This thin band was same as digestion minus band.
There were low concentration band in above of thin band. This thin band was same as digestion minus band.
-
</p>
 
-
 
-
==Transformation==
 
-
<p>
 
-
Transformation of pGEM into BL21. This transformation is the '''plastic production test in BL21'''.
 
-
 
-
#Added 1ul of plasmid DNA to 50ul of thawed competent cells on ice.
 
-
#Incubated on ice for 30min.
 
-
#Added 600ul of LB.
 
-
#Prepared and Labeled two petri dishes with LBA.
 
-
#Plate 300ul of the transformation onto LBA dish and spread.
 
-
#Added 900ul of LB to 100ul of the transformation and plated 300ul of it onto LBA dish then spread.
 
-
#Incubated the plates at 37C for OOhrs.
 
</p>
</p>

Revision as of 03:55, 3 August 2012

Contents

July 30th

digestion

I tried to digest the mini-prep products again, and after Ethanol precipitation, we did electrophoresis.

digestion result

transformation

I think that the E. coli which we use transformation is BL21(DE3)pLysS. So, the E. coli could multiplication increase on LBC plate.
I'm going to do transformation , use DH5α. Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(DH5α).

  1. Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
  2. Incubated on ice for 30min.
  3. Added 200ul of LB then incubated the cells for 2 hours at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Plate 200ul of the transformation onto first dish and spread.
  6. Added 450ul of LB to 50ul of the transformation and plated 200ul of it onto second dish and spread.
  7. Incubated the plates at 37C for 14 hours.

July 31th

liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.

  1. Added 2ml of LBK into culture tubes.
  2. Resuspended colonies.
  3. Incubated the tubes at 37C for

August 1st

mini-prep

mini-prep of pT7-RBS-Ag43-dT which had been cultivated from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions. [[image:|thumb|mini-prep result]]

Digestion

pT7-RBS(20ng/ul) =⑨ SpeI 10xH

DNA solution 4.5ul
SpeI 1ul
10xH buffer 1ul
DW 3.5ul
Total 10ul

SpeI 10xM

DNA solution 4.5ul
SpeI 1ul
10xM buffer 1ul
DW 3.5ul
Total 10ul


pT7-RBS(30ng/ul) =⑩

SpeI 10xH

DNA solution 3ul
SpeI 1ul
10xH buffer 1ul
DW 5ul
Total 10ul

SpeI 10xM

DNA solution 3ul
SpeI 1ul
10xM buffer 1ul
DW 5ul
Total 10ul

[[image:|thumb|digestion result]]

We couldn't cut them exactry so we cut once more time.

pT7-RBS(20ng/ul) =⑨ SpeI 10xH

DNA solution 4.5ul
SpeI 1ul
10xM buffer 2ul
DW 12.5ul
Total 20ul

Liquid culture

Liquid culture for pBAD-RBS on pSB1K3.

  1. Added 2ml of LBK into culture tube.
  2. Scraped the surface of glycerol stock of construct.
  3. Incubated the tube at 33C for OOhrs.

August 2nd

Preparing chemical competent cell

Preparing chemical competent cell of BL21, JM109 and DH5α. Chemical competent cell made in each E.coli strains.

Our competent cell Protocol

  1. Single colony isolation on LB plate
  2. incubated the plate for 15-19 hours at 37℃
  3. lift colony of E. coli into 2 ml LB
  4. cultured cells at 37℃ for 12-16 hours at 180-200 rpm
  5. transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB , respectively
  6. cultured cells at 20℃ (for 24 hours over) at 140 rpm
  7. selected culture by measuring OD 600
  8. left the 300 ml flask for 10 min on ice
  9. transfered the culture into two 50 ml Falcon tube
  10. centrifuged 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
  11. suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer)(7.5 ml/tube)
  12. collected them in one tube
  13. centrifuged 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
  14. suspended the pellet in ice-cold 3.2 ml of TB
  15. Instiled 0.24 ml of DMSO in precipitant
  16. left the 50 ml Falcon tube for 10 min on ice
  17. divide 50ul of solutions in each 0.5 ml tubes
  18. Freezed the suspension in liquid nitrogen
  19. stored at –80℃


Electrophoresis

Electrophoresis of digestion result of yesterday(pT7-RBS on pSB1K3 cut with SpeI)

digestion result

There were low concentration band in above of thin band. This thin band was same as digestion minus band.

Digestion

Digestion of pT7-RBS on pSB1K3(more fresh one)

DNA solution 4 ul
SpeI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


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