IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week5/Chemical and Light

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Ligations 07/22)
(Ligations 07/22)
Line 80: Line 80:
=Ligations 07/22=
=Ligations 07/22=
-
We ligated P1A (vector) to CDF. We used three different ratios of vector to insert for the ligation: 2/6, 1/5, 1/7.
+
*We ligated P1A (vector) to CDF. We used three different ratios of vector to insert for the ligation: 2/6, 1/5, 1/7.
 +
*Attempted to ligate p40 cut with SP (w/ RBS) to mtrB cut with XP.  Thus far, the ligation has not worked.
 +
 
 +
=Recombination Update 07/22=
 +
 
 +
*Thus far, none of the attempts to extract flanking regions from the genomic DNA have worked.  We are troubleshooting this now and will run positive controls with known DNA samples to check the efficiency of our Phusion Polimerase.

Revision as of 14:50, 22 July 2008

Contents

Testing Thermoinducible Lac System

Restreaked Plates of P92-3 in S1 7/21

Restreaked P93 (1:3), P92a, and P93b--all successfully transformed in S1--to detect YFP at 30 and 40 degrees.

Sequencing Parts, Plasmids, Etc.

Primers for Sequencing Remy's Plasmids and Duet Vectors 7/21

Making Thermoinducible cI Lambda System

Primers for Taking Out cI857 from PGW7 7/21

Housekeeping

Making Competent Cells 7/22

PCR

mtrB

7/22: new gradient + R primer

Rx mix (split into 12 samples): 180μL PCR supermix, 4μL mtrB-ApaLI-F primer (20μM), 4μL mtrB-KpnI-R-new (20μM), pipet tip touch of mtrB BB from gel purification, 12μL water Rx: 5min @ 94°C → 35x[45s @ 94°C → 45s @ {52-58 gradient}°C → 2m38s @72°C] → 5min @ 72°C → ∞ @ 4°C

RE digests 07/22

Gel 1

Image:7-22_gel_1_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: CDF PCR cut XP (~900)

Lane 3: CDF PCR uncut (~900)

Lane 4: P1A cut XP (2750)

Lane 5: P1A uncut (3669)

Lane 6: P17 cut EX (5327)

Lane 7: P17 uncut (5327)

Lane 8: P48 cut XP (769)

Lane 9: P48 uncut (3477)


Gel 2

Image:7-22_gel_2_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: P49B cut XP (943)

Lane 3: P49B uncut (3651)

Lane 4: P51 cut EX (5795)

Lane 5: P51 uncut (5795)

Lane 6: P52 cut EX (5412)

Lane 7: P52 uncut (5412)

Lane 8: P63 cut EX (3284)

Lane 9: P63 uncut (3284)


Gel 3

Image:7-22_gel_3_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: P97A cut EX (2091)

Lane 3: P97A uncut (2091)

Lane 4: P97B cut EX (2091)

Lane 5: P97B uncut (2091)

Ligations 07/22

  • We ligated P1A (vector) to CDF. We used three different ratios of vector to insert for the ligation: 2/6, 1/5, 1/7.
  • Attempted to ligate p40 cut with SP (w/ RBS) to mtrB cut with XP. Thus far, the ligation has not worked.

Recombination Update 07/22

  • Thus far, none of the attempts to extract flanking regions from the genomic DNA have worked. We are troubleshooting this now and will run positive controls with known DNA samples to check the efficiency of our Phusion Polimerase.
Personal tools