Team:UCLA/Notebook/Spider Silk Genetics/19 May 2015

iGEM UCLA




5/19/2015

5/19/2015


1 PCR Amplification of MaSp Plasmids

∙ Set up four 25 uL PCR reactions to test amplification. Used two pairs of primers and two different cycling conditions.

∙ Used MaSp2 2CA as template. Diluted 1:1000 to get 219 pg/uL. Created 2 master-mixes, one with each primer pair.

∙ Tested VF2/VR primers and post-elution primers using 17 cycles and 15 cycles using protocol below:

1x Reaction (uL)2x Reaction (uL)
5x Q5 Buffer 5 10
10 mM dNTPs 0.5 1
10 uM For 1.25 2.5
10 uM Rev 1.25 2.5
Template (110 pg)0.5 1
5x GC Buffer 5 10
Q5 Polymerase 0.25 0.5
ddH2O 11.25 22.5
Total 25 50

98 C 30 sec
98 C 10 sec
66 C 15 sec
72 C 15 sec
repeat from step 217x/15x
72 C 2 min
12 C hold

∙ We used 66 C as annealing because that temperature worked for previous amplification with the post-elution primers. In addition, NEB Tm calculator indicates 66 C annealing for the VF2/VR primer pair.

2 Results

∙ Cast 2% TAE gel to visualize results. Used 2 uL of NEB 50 bp ladder.

PIC

Figure 1: P/E indicates reactions with post-elution primer pair. VF/R indicates reactions with VF2 and VR primers. The VF/R primers successfully amplify with a clean band. P/E does not appear to amplify. Primer dimers are present in all lanes. Imaged by Danny. Don’t know why the image is orange.

PIC

Figure 2: P/E indicates reactions with post-elution primer pair. VF/R indicates reactions with VF2 and VR primers. The VF/R primers successfully amplify with a clean band. P/E does not appear to amplify. Primer dimers are present in all lanes. This image taken several hours after previous image. Some band smearing is present.

∙ The VF/R primer pair amplifies our plasmid as expected, but the post-elution primers do not. The expected size for VF/R amplification is 443 bp.

∙ May be due to bad primers for post-elution. Re-order or remake? We need to find more of the post-elution primers.