Team:Colombia Uniandes/NickoJournal

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Revision as of 01:50, 24 July 2013 (view source) Camilog137 (Talk | contribs) (→18-Jun-2013) ← Older edit		Revision as of 01:52, 24 July 2013 (view source) Camilog137 (Talk | contribs) Newer edit →
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	===='''25-Jun-2013'''====		===='''25-Jun-2013'''====
-	So today we took our bacterial cells (Top 10) onto an LB medium (no antibiotics). We let it ON at 37°C on the shaker. Also we prepared all the material we need for tomorrow: ddwater, 10% glycerol, LB medium and microfuge tubes.	+	So today we took our bacterial cells (Top 10) onto an LB medium (no antibiotics). We let it ON at 37 °C on the shaker. Also we prepared all the material we need for tomorrow: ddwater, 10 % glycerol, LB medium and microfuge tubes.
	===='''26-Jun-2013'''====		===='''26-Jun-2013'''====
-	Today we are going to start preparing our cells, we inoculated in the morning and placed all our material that needs to be cold in the fridge 4°C(ddWater and glycerol), also the centifuge rotor needs to be chilled.	+	Today we are going to start preparing our cells, we inoculated in the morning and placed all our material that needs to be cold in the fridge 4 °C(ddWater and glycerol), also the centifuge rotor needs to be chilled.
	Then we just waited for the perfect OD and made our cells around 4:00pm. We made a lot!		Then we just waited for the perfect OD and made our cells around 4:00pm. We made a lot!
Line 26:		Line 26:
	===='''2-Jul-2013'''====		===='''2-Jul-2013'''====
	Our primers are FINALLY here and we are anxious to start working!!!		Our primers are FINALLY here and we are anxious to start working!!!
-	So we made our plan(In theory)on big steps for the next weeks!:	+	So we made our plan(in theory)on big steps for the next weeks!:
	'''1.''' '''DNA extraction''' from ''E.coli'' and ''Ralstonia metallidurans''		'''1.''' '''DNA extraction''' from ''E.coli'' and ''Ralstonia metallidurans''
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	'''3.''' '''Fusion PCR'''		'''3.''' '''Fusion PCR'''
-	3.1 PrcnA/hoxN	+	3.1 PrcnA/''hoxN''
-	3.2 PrcnR,RBS,rcnR/PrcnA/hoxN	+	3.2 PrcnR,RBS,''rcnR''/PrcnA/''hoxN''
	On each PCR add 30 steps more of making gels for confirmation, again and again and again.		On each PCR add 30 steps more of making gels for confirmation, again and again and again.
Line 43:		Line 43:
	===='''3-Jul-2013'''====		===='''3-Jul-2013'''====
-	Fortunately our team member Silvia Cañas already had a DNA extraction from E.coli so she donated it to us so we could start working! Thank you Silvia! :)	+	Fortunately our team member Silvia Cañas already had a DNA extraction from ''E.coli'' so she donated it to us so we could start working! Thank you Silvia! :)
	We asked for ''Ralstonia metallidurans'' to our teacher Jenny Dussan at her lab here in the university. She is giving us the strain tomorrow morning! Thank you Jenny!		We asked for ''Ralstonia metallidurans'' to our teacher Jenny Dussan at her lab here in the university. She is giving us the strain tomorrow morning! Thank you Jenny!
-	This done, did the extraction of ''R.metallidurans'' and store it in the -30°C.	+	This done, did the extraction of ''R.metallidurans'' and store it in the -30 °C.
	===='''4-Jul-2013'''====		===='''4-Jul-2013'''====
-	Today we started with the 2-step PCR of PrcnR, RBS, rcnR, stop (Fragment that will be called "R" from now on).	+	Today we started with the 2-step PCR of PrcnR, RBS, ''rcnR'', stop (Fragment that will be called "R" from now on).
	Results were negative :(		Results were negative :(
Line 61:		Line 61:
	===='''9-Jul-2013'''====		===='''9-Jul-2013'''====
-	So, our PCRs are NOT working....That's why we tried a new procotol of PCR for "R" with a temperature gradient, and we tried new buffers for hoxN.	+	So, our PCRs are NOT working....That's why we tried a new procotol of PCR for "R" with a temperature gradient, and we tried new buffers for ''hoxN''.
	We think our problem is the length of the primers and their constitution that could form secondary structures very VERY easily.		We think our problem is the length of the primers and their constitution that could form secondary structures very VERY easily.
-	We got a POSITIVE result on hoxN, and POSITIVE with "R" at "low" temperatures of annealing. Here is our BEAUTIFULLL gel.	+	We got a POSITIVE result on ''hoxN'', and POSITIVE with "R" at "low" temperatures of annealing. Here is our BEAUTIFULLL gel.
Line 74:		Line 74:
	On 5,7,8,9 we can see "R" fragment of 359bp.		On 5,7,8,9 we can see "R" fragment of 359bp.
-	On 12 we can see hoxN fragment of 837bp.	+	On 12 we can see ''hoxN'' fragment of 837bp.
	===='''11-Jul-2013'''====		===='''11-Jul-2013'''====
Line 82:		Line 82:
	===='''15-Jul-2013'''====		===='''15-Jul-2013'''====
-	We started hoxN PCR with the fusion primers, over the R.metallidurans genome, using 2-step PCR protocol.	+	We started ''hoxN'' PCR with the fusion primers, over the R.metallidurans genome, using 2-step PCR protocol.
	Results are negative.		Results are negative.
Line 88:		Line 88:
	===='''17-Jul-2013'''====		===='''17-Jul-2013'''====
-	We repeated hoxN PCR with the fusion primers over the genome and PrcnA PCR with E.coli genome.	+	We repeated hoxN PCR with the fusion primers over the genome and PrcnA PCR with ''E.coli'' genome.
	Nothing worked OUT :( !!!		Nothing worked OUT :( !!!

---

Revision as of 01:52, 24 July 2013

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Dear Journal! :)

Here we will place all the information about the work we have been doing during this time, it will be named by date. We hope you enjoy our work and thoughts as much as we do!

Our Notebook

18-Jun-2013

Hello! Hello! Hola! Hola! After we went over the project, we proceed to design our primers so we could place our order, taking into account that we are going to use Fusion PCR as our main protocol. Our primers take around 2 weeks to arrive… sooo meanwhilee all the team started making our E.coli babies electrocompetent, so we can work with them later on.

25-Jun-2013

So today we took our bacterial cells (Top 10) onto an LB medium (no antibiotics). We let it ON at 37 °C on the shaker. Also we prepared all the material we need for tomorrow: ddwater, 10 % glycerol, LB medium and microfuge tubes.

26-Jun-2013

Today we are going to start preparing our cells, we inoculated in the morning and placed all our material that needs to be cold in the fridge 4 °C(ddWater and glycerol), also the centifuge rotor needs to be chilled. Then we just waited for the perfect OD and made our cells around 4:00pm. We made a lot!

We placed them in the -80, ready to start our work!!!

2-Jul-2013

Our primers are FINALLY here and we are anxious to start working!!! So we made our plan(in theory)on big steps for the next weeks!:

1. DNA extraction from E.coli and Ralstonia metallidurans

2. PCR

 2.1 PrcnR, RBS, rcnR, stop (Amplified as one piece)
 2.2 PrcnA,RBS
 2.3 hoxN, stop

3. Fusion PCR

 3.1 PrcnA/hoxN
 3.2 PrcnR,RBS,rcnR/PrcnA/hoxN

On each PCR add 30 steps more of making gels for confirmation, again and again and again.

3-Jul-2013

Fortunately our team member Silvia Cañas already had a DNA extraction from E.coli so she donated it to us so we could start working! Thank you Silvia! :)

We asked for Ralstonia metallidurans to our teacher Jenny Dussan at her lab here in the university. She is giving us the strain tomorrow morning! Thank you Jenny!

This done, did the extraction of R.metallidurans and store it in the -30 °C.

4-Jul-2013

Today we started with the 2-step PCR of PrcnR, RBS, rcnR, stop (Fragment that will be called "R" from now on). Results were negative :(

5-Jul-2013

Today we repeated "R" PCR and also did hoxN with the Ralstonia metallidurans genome. Results were negative :( as we could see in our sad, SAD gel.

9-Jul-2013

So, our PCRs are NOT working....That's why we tried a new procotol of PCR for "R" with a temperature gradient, and we tried new buffers for hoxN.

We think our problem is the length of the primers and their constitution that could form secondary structures very VERY easily.

We got a POSITIVE result on hoxN, and POSITIVE with "R" at "low" temperatures of annealing. Here is our BEAUTIFULLL gel.

GoodNewsGEL

On 5,7,8,9 we can see "R" fragment of 359bp. On 12 we can see hoxN fragment of 837bp.

11-Jul-2013

Today we did the PrcnA PCR and it didn't work out.

15-Jul-2013

We started hoxN PCR with the fusion primers, over the R.metallidurans genome, using 2-step PCR protocol.

Results are negative.

17-Jul-2013

We repeated hoxN PCR with the fusion primers over the genome and PrcnA PCR with E.coli genome.

Nothing worked OUT :( !!!

We are going to try to do the PCR of hoxN with the fusion primers over the fragment already amplified and see, as fas as PrcnA goes...we will try a temperature ramp with a 3-step PCR protocol.

End for today :(