Team:Caltech/Week 5

July 11

1 ladder, 2 R0010, 3 K123003, 4 B0014, 5 pSB4A5

Miniprep pSB4A5
Run PCR to amplify R0010, K123003, B0014 and PSB4A5
Run gel of PCR products
Redo PCR of B0014

Results

The PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane.

July 12

from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001

Transformed competent cells with pSB3C5.
Ran PCR of parts for pNT001 and pNT002 Gibson assembly.
Started pulsed field gel electrophoresis of some of our LA River DNA obtained by the Mo Bio kits in week 2.

Results

All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified.

July 13

Retrieved and imaged pulsed field gel.
Ran PCR of R0040 and B0014 with primers that failed yesterday.
Purified PCR products for Gibson of pNT001 and pNT003.
Started overnights of pSB3C5.
Replated some pSB3C5

Results

The pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters.

The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plate and starting overnights to send off for sequencing to make sure the plasmid is okay.
We redid the PCR of R0040 and B0014, but no bands appeared in the gel.

lanes 1 lambda mono cut ladder; 2 blank; 1 fosmid kit control (100 ng); 9-1 Part(2000 ng); 10-1 Part(300 ng); 4 LA River location 9; LA River location 10

PCR Purifications for Gibson

July 14

Miniprep overnights and send off for sequencing
Begin 16S sequencing by PCRing the samples with 16s universal primers and the vector we are using
Try to PCR the miniprep DNA with the pSB3C5 primers
Do Gibson with the PCR parts that worked

Labels for 16s sequencing PCR:

• 9-1 (1/10): 9a
• 9-1 (1/100): 9b
• 9-1 (1/1000): 9c
• 10-3 (1/10): 10a
• 10-3 (1/100): 10b
• 10-3 (1/1000): 10c
• Control 1: C1
• Control 2: C2

Labels for PCR Miniprep:

• pSB3C5 (1/100)- HA
• pSB3C5 (1/100)- HB
• pSB3C5 (1/100)- HC
• pSB3C5 (1/1000)- TA
• pSB3C5 (1/1000)- TB
• pSB3C5 (1/1000)- TC

Results

The minipreps of the overnight cultures all had very low concentrations of DNA.

lane 1 ladder; 2 sample 9; 3 sample 9; 4 sample 9; 5 sample 10; 6 sample 10; 7 sample 10; 8 negative control

lane 1 ladder; 2-7 pSB3C5 minipreps, differing dilutions

ane 1 ladder; 2-4 pET53EST (V1, V2, V3)

July 15

Run gel of LA River samples. Perhaps the freezing and thawing has degraded the DNA further.
Redo PCR of B0014 and R0040 with different annealing temperatures.
Redo Gibson of pNT001 and pNT003, doing 10 ul reactions, transform into Joe's competent cells
Make kanamycin stock and plates.
Gibson assemble 16s with the vector
Transform cells with pSB3K5, because we can't use pSB3C5 in XL-10 Golds

Results

Gibson Plates

Perhaps our cells are not being very competent again. We should do a 20 ul reaction instead of a 10.5 ul reaction so we can retransform if necessary. Also, we should change amounts of DNA in the reaction. We've been diluting the DNA and trying to get all the parts within a 10 ng range.

Lane 1 ladder, 2-4 sample 9, 5-7 sample 10

The LA River DNA has been degraded to be a smear between .1-10kb, too short to use in the pulsed field gel and the fosmid kit.

July 16

Results

We are getting a lot of self-ligation of the vector.
Joe's cells are more competent than ours. We used our XL-10 Golds to transform pSB3K5 and had 0 colonies in the plate. We will need to redo the transformation.

Gibson Plates