Team:University of Lethbridge/Notebook/Project1July

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(July 8, 9)
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====Nathan Puhl, Munima, Christa====
====Nathan Puhl, Munima, Christa====
No colonies on either plate, but the max-efficiency DH5-alpha cells transformed with pSB1A7 were successfully transformed (~1500 cfu).  There are two possibilities: 1. the cells are competent but are low efficiency and we did not add enough DNA, or 2. The cells are not competent.  To assess possibility one we will try more DNA.  If that does not work we will try a protocol using electroporation.
No colonies on either plate, but the max-efficiency DH5-alpha cells transformed with pSB1A7 were successfully transformed (~1500 cfu).  There are two possibilities: 1. the cells are competent but are low efficiency and we did not add enough DNA, or 2. The cells are not competent.  To assess possibility one we will try more DNA.  If that does not work we will try a protocol using electroporation.
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===July 8, 2008===
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====Munima, Christa====
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Objective: Attempt to transform pSB1A7 into RP1616 cells (made competent on July 2/08) again, but using more DNA and changes to June 16/08 transformation protocol
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1. Thawed RP1616 cells and pSB1A7 on ice (obtained from Wieden -80 C freezer)
 +
2. Added 50uL of RP1616 cells to a 15mL falcon tube (prechilled)
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3. Left on ice for 10 minutes
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4. Added 4uL of pSB1A7 to falcon tube
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5. Incubated on ice for 30 minutes
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6. Heat shocked without shaking for 45 seconds at 42 C
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7. Placed on ice for 2 minutes
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8. Added 1mL of SOC media preheated to 42 C. Incubated for 1 hour at 37 C in shaker incubator
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9. Aliquoted 400uL of mixture from incubator into a centrifuge tube
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10. Centrifuged for 1 minute on max speed
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11. Poured off supernatant and resuspended in 100uL of SOC media
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12. Plated 100uL on LB + amp plate. Left overnight in incubator.
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 +
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===July 9, 2008===
 +
====Munima, Christa====
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Growth observed on amp plate that was plated with transformed competent RP1616 cells with pSB1A7. Approximatly 10 colonies were observed.
 +
 +
Subcultured one colony into 5mL LB + amp culture tube (50ug/mL ampicillin). Left overnight in shaker incubator at 37 C at 225 RPM.

Revision as of 23:38, 9 July 2008

Contents

July 1, 2008

Nathan Puhl

Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp


July 2, 2008

Nathan Puhl, Christa, Munima

Plasmid mini prepped pTopp cells

Attempted to make RP1616 cells competent using the open wet ware [http://openwetware.org/wiki/Preparing_chemically_competent_cells protocol]. 100 uL aliquots of cells were frozen in liquid nitrogen and stored in the -80 freezer.

Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year with high range ladder. The pUC19 plasmid seems weird, I don't think it should have two bands; I will have to ask someone about that. The CheZ plasmid appears to be the right size (2600 + 620 = 3220 bp)

Puc19 + cheZ.jpg


July 3, 2008

Nathan Puhl, Munima, Christa

No colonies on either plate, but the max-efficiency DH5-alpha cells transformed with pSB1A7 were successfully transformed (~1500 cfu). There are two possibilities: 1. the cells are competent but are low efficiency and we did not add enough DNA, or 2. The cells are not competent. To assess possibility one we will try more DNA. If that does not work we will try a protocol using electroporation.


July 8, 2008

Munima, Christa

Objective: Attempt to transform pSB1A7 into RP1616 cells (made competent on July 2/08) again, but using more DNA and changes to June 16/08 transformation protocol 

1. Thawed RP1616 cells and pSB1A7 on ice (obtained from Wieden -80 C freezer)
2. Added 50uL of RP1616 cells to a 15mL falcon tube (prechilled)
3. Left on ice for 10 minutes
4. Added 4uL of pSB1A7 to falcon tube
5. Incubated on ice for 30 minutes
6. Heat shocked without shaking for 45 seconds at 42 C
7. Placed on ice for 2 minutes
8. Added 1mL of SOC media preheated to 42 C. Incubated for 1 hour at 37 C in shaker incubator
9. Aliquoted 400uL of mixture from incubator into a centrifuge tube
10. Centrifuged for 1 minute on max speed
11. Poured off supernatant and resuspended in 100uL of SOC media
12. Plated 100uL on LB + amp plate. Left overnight in incubator.


July 9, 2008

Munima, Christa

Growth observed on amp plate that was plated with transformed competent RP1616 cells with pSB1A7. Approximatly 10 colonies were observed.

Subcultured one colony into 5mL LB + amp culture tube (50ug/mL ampicillin). Left overnight in shaker incubator at 37 C at 225 RPM.

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